WO2024044748A2 - Peroxiredoxin 3 inhibitors and methods of use for treating cancer - Google Patents

Peroxiredoxin 3 inhibitors and methods of use for treating cancer Download PDF

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WO2024044748A2
WO2024044748A2 PCT/US2023/072908 US2023072908W WO2024044748A2 WO 2024044748 A2 WO2024044748 A2 WO 2024044748A2 US 2023072908 W US2023072908 W US 2023072908W WO 2024044748 A2 WO2024044748 A2 WO 2024044748A2
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compound
alkyl
mmole
tert
cancer
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PCT/US2023/072908
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French (fr)
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WO2024044748A3 (en
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W. Todd Lowther
Terrence L. SMALLEY, Jr.
Kimberly J. NELSON
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Wake Forest University Health Sciences
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • ROS reactive oxygen species
  • mROS mitochondrial ROS
  • oncogene-induced increases in ROS levels activate the oncogenic transcription factor FOXM1, inducing the expression of FOXM1 target genes including the mitochondrial antioxidant enzymes superoxide dismutase 2 and peroxiredoxin 3 (PRX3).
  • FOXM1 target genes including the mitochondrial antioxidant enzymes superoxide dismutase 2 and peroxiredoxin 3 (PRX3).
  • PRX3 is a peroxidase responsible for metabolizing -90% of mitochondrial hydrogen peroxide (H2O2) (Cox et al. (2009) Biochem J 425, 313-325), and this specific ROS is known to regulate several important processes involved in tumor progression including proliferation, apoptosis, migration and metastasis.
  • the GEPIA2 database of matched pairs of patient samples illustrates how the PRX3 transcript levels are elevated in 15/32 (46.9%) of the tumor tissues collected, including many forms of cancer with significant unmet medical need. Tang et al. (2019) Nucleic Acids Res 47, W556-W560.
  • PRX3 protein expression and mROS levels correlate with sensitivity to the natural product and PRX3 inhibitor thiostrepton (TS) in patient- derived malignant mesothelioma cells lines.
  • TS thiostrepton
  • PRX3 expression supports malignant mesothelioma (MM) and ovarian tumor (OvCa) cell growth. Cunniff et al. (2015) PloS one 10, eO 127310; Myers (2016) Free Radic Biol Med 91, 81- 92; Yoshikawa et al. (2016) Oncol Rep 35, 2543-2552; Wang et al. (2013) Tumour Biol 34, 2275- 2281. PRX3 expression levels in OvCa and cervical cancer also correlate with poor patient outcomes. Li et al. (2016) Biosci Rep 38.
  • PRX3 as a promising molecular target for cancer therapy: (i) no cancer mutations in the PRX3 gene known to support resistance development; (ii) PRX3 KO mice are viable and reach maturity; increase in basal oxidative stress levels observed only in a variety of challenge models (Li et al. (2007) Biochem Biophys Res Commun 355, 715-721; Lee (2020) Antioxidants (Basel) 9); and (iii) partial knockdown of PRX3 via shRNA slows tumor cell proliferation and significantly reduced the expression of FOXM1 at the RNA and protein levels (Cunniff et al. (2015) PloS one 10, e0127310).
  • R 1 is a 5-, 6-, or 7-membered heteroaryl or a heterocycle containing carbon atoms and at least one heteroatom selected from nitrogen and oxygen, which R 1 is substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester and halo; and
  • A is -(CH2)m-, -0-(CH2)m-, or - (CH2)m-0- , wherein m is 0, 1, 2 or 3, or a pharmaceutically acceptable salt or prodrug thereof.
  • R 1 is a 5-membered heteroaryl or heterocycle and the compound is a compound of Formula la: wherein:
  • A is -(CH2)m-, -0-(CH2)m-, or - (CH2)m-0- , wherein m is 0, 1, 2 or 3,
  • X 1 , X 2 and X 3 are each independently N or C;
  • R 2 is an aryl, heteroaryl, cycloalklyl or heterocycle, which R 2 is optionally substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester, and halo.
  • X 1 is N, X 2 is C, and X 3 is N. In some embodiments, X 1 is C, X 2 is N, and X 3 is N. In some embodiments, X 1 is N, X 2 is N, and X 3 is N.
  • R 2 is substituted with one or more selected from alkyl, carboxy, carbamate, urea, amide, and halo. In some embodiments, R 2 is substituted with carbamate or amide. In some embodiments, R 2 is substituted with an alkylcarbamate. In some embodiments, R 2 is a group having a structure of: wherein: n is 0, 1, 2 or 3;
  • Y 1 and Y 2 are each independently absent or is O, NR 6 , or CH2;
  • Z 1 and Z 2 are each independently O, N, or C;
  • R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
  • R 6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • Z 1 and Z 2 are each independently N or C.
  • R 2 is a group having a structure of: wherein: n is 0, 1, 2 or 3;
  • Y 1 is O or CH 2 ;
  • R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
  • R 6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I.
  • R 2 is a group having a structure of: wherein: n is 0, 1, 2 or 3;
  • Y 1 is O or CH2; and R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
  • R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
  • R 2 is a group having a structure of: wherein: n is 0, 1, 2 or 3;
  • Y 1 is O or CH2
  • R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
  • compositions comprising a compound or pharmaceutically acceptable salt or prodrug as taught herein.
  • the composition is formulated for oral or parenteral (e.g. intravenous, intrapleural, intraperitoneal or intraovarian) administration.
  • the composition is formulated for oral administration and is in the form of a capsule, cachet, lozenge, or tablet.
  • the formulation is provided in unit dosage form of from 1 mg to 10 grams of the compound, pharmaceutically acceptable salt or prodrug.
  • a method treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof.
  • a compound of Formula I or a pharmaceutically acceptable salt or prodrug thereof for use in treating cancer in a subject in need thereof, or for preparing a medicament for use in treating cancer.
  • the cancer has PRX3 expression.
  • the subject is a human subject. In some embodiments, the subject is a non-human animal subject (e.g. non-human mammalian subject).
  • the administering is carried out by administering a pharmaceutical composition comprising said compound or pharmaceutically acceptable salt or prodrug.
  • the administering further comprises administering bortezomib, carboplatin, paclitaxel, an immunotherapy agent, or a combination thereof. In some embodiments, the administering further comprises administering doxorubicin. Further provided is a method of inhibiting PRX3 in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof.
  • H refers to a hydrogen atom.
  • C refers to a carbon atom.
  • N refers to a nitrogen atom.
  • S refers to a sulfur atom.
  • O refers to an oxygen atom.
  • Alkyl refers to a saturated straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms.
  • Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3 -methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n- nonyl, n-decyl, and the like.
  • Lower alkyl as used herein, is a subset of alkyl and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms.
  • Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like.
  • the alkyl groups may be optionally substituted with one or more suitable substituents, such as halo, hydroxy, carboxy, amine, etc.
  • Cycloalkyl refers to a saturated cyclic hydrocarbon containing from 1 to 10 carbon atoms.
  • Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
  • the cycloalkyl groups may be optionally substituted with one or more suitable substituents, such as halo, hydroxy, carboxy, amine, etc.
  • Aryl refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused or directly adjoining ring system having one or more aromatic rings. Examples include, but are not limited to, phenyl, indanyl, indenyl, tetrahydronaphthyl, biphenyl, napthyl, azulenyl, etc.
  • the aryl may be optionally substituted with one or more suitable substituents, such as alkyl, halo, hydroxy, carboxy, amine, etc.
  • Heteroaryl refers to a monovalent aromatic group having a single ring or two fused or directly adjoining rings and containing in at least one of the rings at least one heteroatom (typically 1 to 3) independently selected from nitrogen, oxygen and sulfur.
  • Examples include, but are not limited to, pyrrole, imidazole, thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, benzothiophene, benzofuran, indole, benzimidazole, benzothiazole, quinoline, isoquinoline, quinazoline, quinoxaline, phenyl -pyrrole, phenyl-thiophene, etc.
  • the heteroaryl may be optionally substituted with one or more suitable substituents, such as alkyl, halo, hydroxy, carboxy, amine, etc.
  • Heterocycle refers to a saturated or partially unsaturated cyclic hydrocarbon with at least one heteroatom (typically 1 to 3) independently selected from nitrogen, oxygen and sulfur.
  • the heterocycle may be a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle.
  • the heterocycle may be optionally substituted with one or more suitable substituents, such as alkyl, halo, hydroxy, carboxy, amine, etc.
  • “Monocyclic heterocycle” means a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom, and which is not aromatic.
  • Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3- dioxolanyl, dihydropyranyl (including 3, 4-dihydro-2H-pyran-6-yl), 1,3-dithiolanyl, 1,3-dithianyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxadiazolidinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrrolidinyl, tetrahydrofuranyl, tetra
  • Bicyclic heterocycle means a monocyclic heterocycle fused to an aryl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle.
  • bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3 -benzodi oxolyl, 1,3-benzodithiolyl, 2,3-dihydro-l,4-benzodioxinyl, 2,3 -dihydro-1 -benzofuranyl, 2, 3-dihy dro-1 -benzothienyl, 2,3- dihydro-lH-indolyl, 3,4-dihydroquinolin-2(lH)-one and 1,2, 3, 4- tetrahydroquinolinyl.
  • Tricyclic heterocycle means a bicyclic heterocycle fused to an aryl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle.
  • Representative examples of tricyclic heterocycles include, but are not limited to, 2,3,4,4a,9,9a-hexahydro- IH-carbazolyl, 5a,6,7,8,9,9a-hexahydro- dibenzo[b,d] furanyl, and 5a,6,7,8,9,9a-hexahydrodibenzo[b,d]thienyl.
  • halo and "halogen,” as used herein, refer to fluoro (-F), choro (-C1), bromo (- Br), or iodo (-1).
  • Haloalkyl refers to one or more halo groups appended to the parent molecular moiety through an alkyl group. Examples include, but are not limited to, chloromethyl, fluoromethyl, trifluoromethyl, etc.
  • Carboxy refers to the group -COOH.
  • Alkoxy refers to an alkyl or cycloalkyl group, as herein defined, attached to the principal carbon chain through an oxygen atom.
  • Representative examples of “alkoxy” include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, and hexyloxy.
  • amine or “amino” refers to a group -NH2, wherein none, one or two of the hydrogens may be replaced by an alkyl, cycloalkyl, or aryl as defined herein.
  • amide refers to a group having a carbonyl bonded to a nitrogen atom, such as -C(0)NH2, wherein none, one or two of the hydrogens may be replaced by an alkyl, cycloalkyl, or aryl as defined herein.
  • ether refers to a group in which there is an ether, R-O-R', wherein R and R' are each independently an alkyl, cycloalkyl, or aryl as defined herein.
  • esters refers to a group in which there is an ester, R-C(O)-O-R', wherein R and R' are each independently an alkyl, cycloalkyl, or aryl as defined herein.
  • a “carbamate” refers to a group in which there is a carbamate, R-O-C(O)NR'R", wherein R, R' and R" are each independently an alkyl, cycloalkyl, or aryl as defined herein.
  • a “urea” refers to a group in which there is a urea, R-NH-C(O)-NH-R', wherein R and R' are each independently an alkyl, cycloalkyl, or aryl as defined herein.
  • substituted indicates that the specified group is either unsubstituted, or substituted by one or more suitable substituents.
  • a "substituent” that is “substituted” is a group which takes the place of one or more hydrogen atoms on the parent organic molecule.
  • salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.
  • examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine.
  • Active compounds useful as PRX3 inhibitors in accordance with the present invention are provided below.
  • structures depicted herein are also meant to include all enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention.
  • all tautomeric forms of the compounds of the invention are within the scope of the invention. Tautomeric forms include keto-enol tautomers of a compound.
  • all rotamer forms of the compounds of the invention are within the scope of the invention.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention.
  • Such compounds are useful, for example, as analytical tools or probes in biological assays.
  • an active compound is a compound of Formula I: wherein R 1 is a 5-, 6-, or 7-membered heteroaryl or a heterocycle containing carbon atoms and at least one heteroatom selected from nitrogen and oxygen, which R 1 is substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester and halo; and
  • A is -(CH2)m-, -0-(CH2)m-, or - (CH2)m-0- , wherein m is 0, 1, 2 or 3, or a pharmaceutically acceptable salt or prodrug thereof.
  • R 1 is a 5-membered heteroaryl or heterocycle and the compound is a compound of Formula la: wherein:
  • A is as defined in claim 1;
  • X 1 , X 2 and X 3 are each independently N or C;
  • R 2 is an aryl, heteroaryl, cycloalklyl or heterocycle, which R 2 is optionally substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester, and halo.
  • X 1 is N, X 2 is C, and X 3 is N. In some embodiments, X 1 is C, X 2 is N, and X 3 is N. In some embodiments, X 1 is N, X 2 is N, and X 3 is N.
  • R 2 is substituted with one or more selected from alkyl, carboxy, carbamate, urea, amide, and halo. In some embodiments, R 2 is substituted with carbamate or amide. In some embodiments, R 2 is substituted with an alkylcarbamate.
  • R 2 is a group having a structure of: wherein: n is 0, 1, 2 or 3;
  • Y 1 and Y 2 are each independently absent or is O, NR 6 , or CH2;
  • Z 1 and Z 2 are each independently O, N, or C;
  • R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
  • R 6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • Z 1 and Z 2 are each independently N or C.
  • R 2 is a group having a structure of: wherein: n is 0, 1, 2 or 3;
  • Y 1 is O or CH 2 ;
  • R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
  • R 6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I.
  • R 2 is a group having a structure of: wherein: n is 0, 1, 2 or 3;
  • Y 1 is O or CH2; and R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
  • R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
  • R 2 is a group having a structure of: wherein: n is 0, 1, 2 or 3;
  • Y 1 is O or CH2
  • R 5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
  • active compounds include, but are not limited to, those selected from
  • an active compound can form a covalent adduct with PRX3 in a biochemical PRX3 inhibition assay, which may support its PRX3 inhibition activity.
  • an active compound can have an ECso in a cellular activity assay (such as the ability to kill cancer cells such as SKOV3 ovarian cancer cells) in the micromolar range, such as from 0.05, 0.1, 0.25, or 0.5 micromolar, to 10, 15, or 20 micromolar.
  • an active compound can have good solubility, e.g., solubility in aqueous solution (e.g., saline such as phosphate buffered saline, water, etc.) of at least 0.1 millimolar, such as from 0.1 to 1 millimolar, or solubility in an aqueous solution of at least 1 millimolar.
  • solubility in aqueous solution e.g., saline such as phosphate buffered saline, water, etc.
  • an active compound can have good aqueous stability.
  • an active compound may have no decrease in purity after 24 hours in an aqueous solution.
  • an active compound does not have appreciable antimicrobial activity, e.g., at greater than 20 micromolar concentrations.
  • an active compound does not inhibit the proteasome and/or does not inhibit FOXM1 DNA binding. These may indicate that the compound has greater specificity for PRX3 than TS.
  • treat refers to any type of treatment that imparts a benefit to a subject afflicted with a disease or disorder, delay in the progression of the disease or disorder, or symptoms thereof, etc.
  • the treatment is for a cancer (e.g., a cancer having elevated reactive oxygen species).
  • the subject treated is a human subject.
  • the subject is a non-human animal (e.g., non-human mammalian subject).
  • a non-human animal may include, but is not limited to, non-human primates, dogs, cats, horses, cattle, goats, pigs, sheep, guinea pigs, mice, rats and rabbits, as well as any other domestic, commercially or clinically valuable animal, including but not limited to animal models and livestock animals.
  • the subject is a subject in need of a treatment such as a treatment of the present invention.
  • Cancers that may be treated with the active compounds according to some embodiments may include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bile duct cancer; bladder cancer; bone cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cardiac
  • liver cancer e.g., hepatocellular cancer (HCC), malignant hepatoma
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung
  • leiomyosarcoma LMS
  • mastocytosis e.g., systemic mastocytosis
  • melanoma midline tract carcinoma; multiple endocrine neoplasia syndrome; muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.
  • MMD myeloproliferative disorder
  • myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); nasopharynx cancer; neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors
  • the cancer is a blood cancer such as leukemia, liver cancer, lung cancer, lymphoma, melanoma, prostate cancer, head and neck cancer, bladder cancer, brain cancer, breast cancer, or cervical cancer.
  • the cancer is prostate cancer.
  • the cancer is head and neck cancer.
  • the cancer is ovarian cancer.
  • the cancer is cervical cancer.
  • the cancer is malignant mesothelioma.
  • the cancer has PRX3 expression.
  • the cancer may be a cancer type generally known to express PRX3 and/or the cancer has been determined (e.g., by testing a biopsy) to have PRX3 expression.
  • the cancer may be metastatic, in which cancerous cells from a primary or original tumor migrate to another organ or tissue and may be identified as the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located.
  • a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.
  • the active compounds disclosed herein can, as noted above, be prepared in the form of their pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.
  • Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid,
  • Active compounds of the present invention may be prepared as pharmaceutically acceptable prodrugs.
  • Such prodrugs are those which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable risk/benefit ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
  • the term "prodrug” refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood.
  • Examples include a prodrug that is metabolized in vivo by a subject to an active drug having an activity of active compounds as described herein, wherein the prodrug is an ester of an alcohol or carboxylic acid group, if such a group is present in the compound; an acetal or ketal of an alcohol group, if such a group is present in the compound; an N-Mannich base or an imine of an amine group, if such a group is present in the compound; or a Schiff base, oxime, acetal, enol ester, oxazolidine, or thiazolidine of a carbonyl group, if such a group is present in the compound, such as described in US Patent No. 6,680,324 and US Patent No. 6,680,322.
  • the active compounds described above may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (9th Ed. 1995).
  • the active compound (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier.
  • the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient.
  • the carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight of the active compound.
  • One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy comprising admixing the components, optionally including one or more accessory ingredients.
  • compositions of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature, severity and location of the condition being treated and on the nature of the particular active compound which is being used.
  • Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
  • Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above).
  • the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
  • a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s).
  • Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
  • Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
  • Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound(s), which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents.
  • the formulations may be presented in unit/dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.
  • sterile liquid carrier for example, saline or water-for-inj ection immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • an injectable, stable, sterile composition comprising an active compound(s), or a salt thereof, in a unit dosage form in a sealed container.
  • the compound or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject.
  • the unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt.
  • a sufficient amount of emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier.
  • emulsifying agent is phosphatidyl choline.
  • Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
  • Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.
  • Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.
  • the present invention provides liposomal formulations of the compounds disclosed herein and salts thereof.
  • the technology for forming liposomal suspensions is well known in the art.
  • the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes.
  • the lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol-free.
  • the salt When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques.
  • the liposomal formulations containing the compounds disclosed herein or salts thereof may be lyophilized to produce a lyophilizate which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
  • compositions may be prepared from the compounds disclosed herein, or salts thereof, such as aqueous base emulsions.
  • the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the compound or salt thereof.
  • Particularly useful emulsifying agents include phosphatidyl cholines, and lecithin.
  • the pharmaceutical compositions may contain other additives, such as pH-adjusting additives.
  • useful pH-adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate.
  • the compositions may contain microbial preservatives.
  • Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use. If desired, the pharmaceutical compositions of the present invention may be lyophilized using techniques well known in the art. V. Dosage and routes of administration.
  • the present invention provides pharmaceutical formulations comprising the active compounds (including the pharmaceutically acceptable salts thereof), in pharmaceutically acceptable carriers for oral, rectal, topical, buccal, parenteral, intrapleural, intraovarian, intramuscular, intradermal, intravascular, and/or transdermal administration.
  • Parenteral administration may be, for example, intravascular (intravenous or intraarterial), intrapleural, intraperitoneal or intraovarian administration by injection, infusion or implantation.
  • the therapeutically effective dosage of any specific compound will vary somewhat from compound to compound, and patient to patient, and will depend upon the condition of the patient and the route of delivery.
  • a dosage from about 0.1 to about 50 mg/kg is expected to have therapeutic efficacy, with all weights being calculated based upon the weight of the active compound, including the cases where a salt is employed.
  • Toxicity concerns at the higher level may restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the active base, including the cases where a salt is employed.
  • a dosage from about 10 mg/kg to about 50 mg/kg may be employed for oral administration.
  • a dosage from about 0.5 mg/kg to 5 mg/kg may be employed for intramuscular injection.
  • the compounds described herein may be administered alone or concurrently with one or more additional active agent useful for treating the disease or condition with which the patient is afflicted.
  • additional active agents include, but are not limited to, those set forth in paragraphs 0065 through 0387 of W. Hunter, D. Gravett, et al., US Patent Application Publication No. 20050181977 (Published August 18, 2005) (assigned to Angiotech International AG) the disclosure of which is incorporated by reference herein in its entirety.
  • Methyl ⁇ 9-(tert-butyldimethylsilyl)-7V-(l -phenyl- I H-pyrazole-4-carbonyl )-/.-seryl-/.-serinate (0.167 g, 0.340 mmole) was dissolved in DCM (2 mL) and cooled to 0°C. Triethylamine (0.072 mL, 0.514 mmole) was added followed by methanesulfonyl chloride (0.040 mL, 0.517 mmole). The solution was stirred at 0° for 2 hours and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL).
  • Methyl (5)-2-(3-((tert-butyldimethylsilyl)oxy)-2-(l -phenyl- lH-pyrazole-4- carboxamido)propanamido)acrylate (0.175 g, 0.370 mmole) was dissolved in THF (2 mL) and tetrabutylammonium fluoride (IM solution in THF, 0.410 mL, 0.410 mmole) was added. The solution was stirred at RT for 20 hours and water (25 mL) was added.
  • IM solution in THF 0.410 mL, 0.410 mmole
  • Methyl (S)-2-(3-hydroxy-2-(l-phenyl-lH-pyrazole-4-carboxamido)propanamido)acrylate (0.143 g, 0.399 mmole) was dissolved in DCM (2 mL) and cooled to 0°C. Triethylamine (0.084 mL, 0.599 mmole) was added followed by methanesulfonyl chloride (0.046 mL, 0.594 mmole) and the mixture was stirred at 0° for 2 hours. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated.
  • tert-butyl 4-(((((2,5-dioxopyrrolidin-l- yl)oxy)carbonyl)oxy)methyl)piperidine-l -carboxylate as an orange oil (1.972 g) that was carried on without additional purification.
  • T ert-butyl (5)-4-(((3 -hydroxy- 1 -methoxy- 1 -oxopropan-2-yl)carbamoyl)oxy)piperidine- 1 - carboxylate (1.526 g, 4.41 mmole) was dissolved in DCM (8 mL). Imidazole (0.329 g, 4.83 mmole) was added followed by tert-butyldimethylchlorosilane (0.736 g, 4.88 mmole). The mixture was stirred at RT for 60 minutes and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated.
  • the crude residue was purified by silica gel chromatography to provide tert-butyl 4-(((3 -((3 -m ethoxy-3 -oxoprop- l-en-2-yl)amino)- 3 -oxoprop- l-en-2-yl)carbamoyl)oxy)piperidine-l -carboxylate as a thick, pale yellow oil (0.215 g, 44%).
  • tert-butyl 4-azidopiperidine-l -carboxylate as a colorless oil (1.657 g, 74%) and the intermediate tert-butyl 4-((methylsulfonyl)oxy)piperidine-l-carboxylate as a white solid (0.231 g, 8%).
  • tertbutyl 4-hydroxypiperidine-l -carboxylate (2.032 g, 10.1 mmole) was dissolved in DCM (20 mL) and cooled to 0°C. Triethylamine (1.55 mL, 11.1 mmole) was added followed by methanesulfonyl chloride (0.850 mL, 11.0 mmole). The mixture was stirred at 0° for 2 hours and water (50 mL) was added. The two layers were separated and the aqueous layer was extracted with DCM (2 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated.
  • the crude residue was purified by silica gel chromatography (20-50% ethyl acetate/hexane gradient) to provide tert-butyl 4-(3- (methoxycarbonyl)-lH-pyrazol-l-yl)piperidine-l-carboxylate as a colorless oil (0.582 g, 19%).
  • the crude residue was purified by silica gel chromatography (5-30% ethyl acetate/hexane gradient) to provide tert-butyl (5)-4-(3-((3-((tert-butyldimethylsilyl)oxy)-l-methoxy-l-oxopropan-2- yl)carbamoyl)-U/-pyrazol-l-yl)piperidine-l -carboxylate as a colorless oil (0.636 g, 66%).
  • the crude residue was purified by silica gel chromatography (30-60% ethyl acetate/hexane gradient) to provide tert-butyl 4-(3-(((65,95)-9- (m ethoxy carbonyl)-2, 2, 3, 3 -tetramethyl-7,12-di oxo-4, 11 -di oxa-8-aza-3 -silatridecan-6- yl)carbamoyl)-17/-pyrazol-l -yl)piperidine-l -carboxylate as a colorless oil (0.463 g, 81%).
  • the crude residue was purified by silica gel chromatography (10-50% ethyl acetate/hexane gradient) to provide tert-butyl 4-(3 -((3 -((3 -methoxy-3 -oxoprop- 1 -en-2-yl)amino)-3 -oxoprop- 1 -en-2- yl)carbamoyl)-U/-pyrazol-l-yl)piperidine-l -carboxylate as a white solid (0.087 g, 27%).
  • 6-Bromohexanoic acid (1.964 g, 10.1 mmole) was dissolved in DCM (20 mL) and DMF (1 drop) was added.
  • Oxalyl chloride (0.880 mL, 10.1 mmole) was added dropwise and the solution was stirred at RT for 90 minutes, then was concentrated. The residue was dissolved in DCM (2 mL) and added dropwise to cold (0°C) solution of 4-(4, 4, 5, 5-tetramethyl-l, 3, 2-dioxaborolan-2- yl)aniline (2.007 g, 9.16 mmole) and N,N-diisopropylethylamine (3.20 mL, 18.4 mmole) in DCM (20 mL).
  • the crude residue was purified by silica gel chromatography (Isco CombiPrep, 24 g RediSep column, 20-50% ethyl acetate/hexane gradient) to provide methyl N- (2-(4-(6-bromohexanamido)phenyl)thiazole-4-carbonyl)-O-(tert-butyldimethylsilyl)-L-serinate as a thick, orange gel (1.290 g, 44%).
  • the compounds described herein generally react with two essential cysteine residues in the mitochondrial peroxidase PRX3, leading to the formation of a non-reducible, covalent crosslink of two PRX3 monomers. This crosslink inactivates PRX3. Increased PRX3 crosslink is associated with mitochondrial stress, increased cell death in cell models of malignant mesothelioma and is associated with decreased tumor volume in a mouse model. Cunniff et al. (2015) PloS one 10, e0127310.
  • PRX3 uses an essential reduced cysteine residue to reduce hydrogen peroxide. During this process, PRX3 becomes oxidized forming a reversible disulfide bond that links two PRX3 monomers. In the cell, this disulfide can be reduced by the combined activity of thioredoxin 2 (TRX2), thioredoxin reductase 2, and NADPH. The disulfide can also be reduced by small molecule reductants such as dithiolthreitol (DTT). In contrast, the compound-crosslink with PRX3 is irreversible and cannot be broken by the addition of reductants.
  • TRX2 thioredoxin 2
  • DTT dithiolthreitol
  • the Biochemical PRX3 Inhibition Assay tests the ability of each compound to crosslink PRX3 in a simple, in vitro system.
  • the Cellular Activity Assay we are testing the ability of compounds to kill SK-OV-3 ovarian cancer cells.
  • This assay tests the ability of each compound to form a covalent adduct with PRX3.
  • the assay is performed as described in Nelson et al. (2021) Antioxidants (Basel) 10, 150; and Cunniff et al. (2015) PloS one 10, e0127310, and follows the appearance of non-reducible PRX3 crosslinks by SDS PAGE gel electrophoresis and follows the appearance of single PRX3 adducts by mass spectrometry.
  • purified human PRX3, hydrogen peroxide (the PRX3 substrate) and all the components required to enable PRX3 to catalytically cycle are included (details below).
  • the biochemical PRX3 Inhibition assay contains 100 pM PRX3, 50 pM human TRX2, 0.5 pM mouse thioredoxin reductase, and a NADPH regenerating system composed of 3.2 mM glucose 6-phosphate, 3.2 U/ml glucose 6-phosphate dehydrogenase and 0.4 mM NADPH. Samples are incubated for 1-2 hr at 37°C with either 0.2 mM TS (positive control), compounds or an equivalent volume of DMSO (negative control). During this incubation, hydrogen peroxide is added to induce turnover of PRX3.
  • Reactions are stopped by the addition of a buffer containing 100 mM dithiolthreitol (to break disulfide bonds) and SDS (detergent to denature proteins).
  • NADPH, Glucose 6-Phosphate, and glucose 6-phosphate dehydrogenase were purchased from Sigma Aldrich.
  • PRX3, thioredoxin, and thioredoxin reductase were all purified to >98% purity in the Lowther laboratory according to protocols referenced in Nelson et al. (2021) Antioxidants (Basel) 10, 150; and Cunniff et al. (2015) PloS one 10, e0127310.
  • PRX3 crosslink proteins in the reaction were separated by SDS- polyacrlyamide gel electrophoresis and stained for total protein using GelCode Blue (Life Technologies). The amount of unmodified PRX3 and TS-PRX3 crosslink was measured by densiometric analysis of the signal for the PRX3 band running at the MW of a PRX3 crosslink ( ⁇ 46 kDa) compared to the PRX3 signal at the MW of the un-modified PRX3 ( ⁇ 23 kD).
  • each reaction was exchanged into a mass spectrometry compatible buffer containing 40 mM ammonium citrate, pH 8.3 made in HPLC water.
  • Sample was mixed 1 : 1 with a matrix solution containing 30 mg/mL sinapinic acid in 70% (vol/vol) acetonitrile, 0.2% formic acid and spotted to onto the sample plate.
  • PRX3 mass was measured by MALDLTOF MS analysis on a Bruker Daltonics MALDLTOF MS spectrometer. Spectra were analyzed in FLEXAnalysis Software.
  • the intensity of the reduced PRX3 peak (SH) and intensity of the analog adduct peak was determined and corrected for background signal at the adduct peak in the DMSO control.
  • the fraction of single analog adduct in the monomer peak was determined by dividing the intensity of the adduct peak by the summed intensity of the SH and adduct peaks.
  • the Cellular Activity Assay measures the ECso of each compound in human SK-OV-3 ovarian cancer cells TS to be taken into the cell, transported to the mitochondria, and crosslink PRX3.
  • SK-OV-3 is an adherent, epithelial, adenocarcenoma cell line obtained from ATCC (ref #: HTB-77). SK-OV-3 cells are resistant to the commonly used chemotherapeutics, cis-platinum and doxorubicin. For this PRX3 crosslinking assay, SK-OV-3 cells were plated in a 96-well plate. After 24 h recovery, the cells were treated for 48 hr with multiple concentrations of each compound ranging from 0.1 - 100 pM.
  • Crystal violet was determined by reading the absorbance at 540 nm (crystal violet dye dissolved in 100% methanol) using a plate reader. The signal for crystal violet dye is proportional to the biomass of remaining live cells.
  • GraphPad Prism9 software was used to calculate the effective inhibitory concentration (ECso) of test compounds. Results for each compound, are normalized to the amount of cells in control wells treated with the equivalent concentration of DMSO (negative control). TS or Compound A (Reference Compound) were included in each set of assays as a positive control.
  • MIC minimal inhibitory concentration
  • TS with Enterococcus hirae ATCC # 10541
  • BHI growth media 7.8 g/L brain extract, 2.0 g/L dextrose, 2.5 g/L di sodium phosphate, 9.7 g/L heart extract, 10 g/L proteose peptone, 5 g/L sodium chloride supplemented with 0.01% (v/v) polysorbate 80, 10 g/mL dextrose, and 10 g/L agar.
  • BHI growth media 7.8 g/L brain extract, 2.0 g/L dextrose, 2.5 g/L di sodium phosphate, 9.7 g/L heart extract, 10 g/L proteose peptone, 5 g/L sodium chloride supplemented with 0.01% (v/v) polysorbate 80, 10 g/mL dextrose, and 10 g/L agar.
  • hirae was treated with 20 pM final concentration of each analog (three replicates each). No growth inhibition was observed for any analog.
  • DMSO was used as a positive control (complete cell growth) and 20 pM thiostrepton was used as a negative control.
  • GEPIA2 an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic Acids Res 47, W556- W560
  • Peroxiredoxin 3 is a redox-dependent target of thiostrepton in malignant mesothelioma cells.

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Abstract

Provided according to some embodiments is a compound of Formula I, or a pharmaceutically acceptable salt thereof. Pharmaceutical compositions comprising the same and methods of use for treating cancer and inhibiting PRX3 are also provided.

Description

PEROXIREDOXIN 3 INHIBITORS AND METHODS OF USE
FOR TREATING CANCER
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under Contract Number R01 GM072866 awarded by the National Institutes of Health. The U.S government has certain rights in the invention.
BACKGROUND
A central hallmark of the tumorigeneses of cells is metabolic changes that cause an increase in the level of reactive oxygen species (ROS) and mitochondrial ROS (mROS). Cairns et al. (2011) Nat Rev Cancer 11, 85-95; Weinberg et al. (2009) Cell Mol Life Sci 66, 3663-3673; Weinberg et al. (2009) Ann N Y Acad Sci 1177, 66-73; Weinberg et al. (2010) Proc Natl Acad Sci USA 107, 8788-8793. In response to this neoplastic transformation, cells reorganize their antioxidant capacity to survive, proliferate and metastasize. Specifically, oncogene-induced increases in ROS levels activate the oncogenic transcription factor FOXM1, inducing the expression of FOXM1 target genes including the mitochondrial antioxidant enzymes superoxide dismutase 2 and peroxiredoxin 3 (PRX3). Park et al. (2009)EA7BG J28, 2908-2918; Nonn et al. (2003)Afo/ Cancer Res 1, 682-689.
PRX3 is a peroxidase responsible for metabolizing -90% of mitochondrial hydrogen peroxide (H2O2) (Cox et al. (2009) Biochem J 425, 313-325), and this specific ROS is known to regulate several important processes involved in tumor progression including proliferation, apoptosis, migration and metastasis. The GEPIA2 database of matched pairs of patient samples (tumor vs. normal) illustrates how the PRX3 transcript levels are elevated in 15/32 (46.9%) of the tumor tissues collected, including many forms of cancer with significant unmet medical need. Tang et al. (2019) Nucleic Acids Res 47, W556-W560. PRX3 protein expression and mROS levels correlate with sensitivity to the natural product and PRX3 inhibitor thiostrepton (TS) in patient- derived malignant mesothelioma cells lines. Nelson et al. (2Q2Y) Antioxidants (Basel) 10, 150.
PRX3 expression supports malignant mesothelioma (MM) and ovarian tumor (OvCa) cell growth. Cunniff et al. (2015) PloS one 10, eO 127310; Myers (2016) Free Radic Biol Med 91, 81- 92; Yoshikawa et al. (2016) Oncol Rep 35, 2543-2552; Wang et al. (2013) Tumour Biol 34, 2275- 2281. PRX3 expression levels in OvCa and cervical cancer also correlate with poor patient outcomes. Li et al. (2018) Biosci Rep 38. The following additional features further support PRX3 as a promising molecular target for cancer therapy: (i) no cancer mutations in the PRX3 gene known to support resistance development; (ii) PRX3 KO mice are viable and reach maturity; increase in basal oxidative stress levels observed only in a variety of challenge models (Li et al. (2007) Biochem Biophys Res Commun 355, 715-721; Lee (2020) Antioxidants (Basel) 9); and (iii) partial knockdown of PRX3 via shRNA slows tumor cell proliferation and significantly reduced the expression of FOXM1 at the RNA and protein levels (Cunniff et al. (2015) PloS one 10, e0127310).
A study by Corsello et al. (Nature Cancer 2020 l(2):235-248) tested the ability of 4,518 drugs from the Drug Repurposing Hub at the Broad Institute to kill 578 cancer cells lines. Thiostrepton (TS), an insoluble, thiopeptide antibiotic, showed meaningful efficacy in 403 tumor cells lines derived from a wide array of tissues. Our team has demonstrated that TS acts by irreversibly crosslinking the two essential catalytic cysteine residues in PRX3, inactivating peroxidase activity and increasing ROS to levels incompatible with survival. Nelson et al. (2021) Antioxidants (Basel) 10, 150; Cunniff et al. (2015) PloS one 10, e0127310; Newick et al. (2012) PloS one 7, e39404. Because this irreversible crosslink occurs across the homodimer interface, the inactivated PRX3 is significantly larger in mass, and we can track the PRX3 crosslink in our cellular and animal models.
Several mechanisms have been proposed for TS cytotoxicity of cancer cells: (i) interaction with the oncogenic transcription factor FOXM1 (Hegde et al. (2011) Nat Chem 3, 725-731) (ii) inhibition of the 20/26S proteasome (Bhat et al. (2009) PloS one 4, e6593; Bird et al. (2020) ACS Chem Biol 15, 2164-2174), (iii) binding to the large subunit of ribosomes (Zhang et al. (2005) Antibiotic susceptibility of mammalian mitochondrial translation. FEBS Lett 579, 6423-6427; Harms et al. (2008) Mol Cell 30, 26-38), and (iv) covalent adduction and cross-linking of PRX3 by our team (Nelson et al.
Figure imgf000003_0001
Antioxidants (Basel) 10, 150; Cunniff et al. (2015) PloS one 10, e0127310). We have shown that TS sensitivity is greatly decreased upon knockdown of PRX3 in a cell model of MM, indicating that PRX3 inhibition is key in driving TS cytotoxicity. Inhibition of PRX3 also significantly increases mitochondrial ROS which drives TS-mediated cell death. Increased ROS modulates FOXM1 expression while increased production of mitochondrial ROS has also been shown to disassemble 26S proteasome complexes (Livnat-Levanon et al. (2014) Cell Rep 7, 1371-1380; Segref et al. (2014) Cell Metab 19, 642-652), further complicating the interpretation of the mode of action of TS.
Despite the effectiveness of TS in cell and animal models of cancer, this natural product has serious limitations to its utility as a chemotherapy. First, it is highly large, highly insoluble, and does not exhibit any of the preferred drug like properties. Second, this molecule is currently produced by bacterial fermentation followed by organic extraction and purification. Although a synthetic route for synthesis has been published, it involved multiple steps, is expensive, and has low yield. Ayida et al. (2005) Bioorg Med Chem Lett 15, 2457-2460.
Improved PRX3 inhibitors that can address some of these issues are needed.
SUMMARY
Provided herein according to some embodiments is a compound of Formula I:
Figure imgf000004_0001
wherein R1 is a 5-, 6-, or 7-membered heteroaryl or a heterocycle containing carbon atoms and at least one heteroatom selected from nitrogen and oxygen, which R1 is substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester and halo; and
A is -(CH2)m-, -0-(CH2)m-, or - (CH2)m-0- , wherein m is 0, 1, 2 or 3, or a pharmaceutically acceptable salt or prodrug thereof.
In some embodiments, R1 is a 5-membered heteroaryl or heterocycle and the compound is a compound of Formula la:
Figure imgf000004_0002
wherein:
A is -(CH2)m-, -0-(CH2)m-, or - (CH2)m-0- , wherein m is 0, 1, 2 or 3,
X1, X2 and X3 are each independently N or C; and
R2 is an aryl, heteroaryl, cycloalklyl or heterocycle, which R2 is optionally substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester, and halo.
In some embodiments, X1 is N, X2 is C, and X3 is N. In some embodiments, X1 is C, X2 is N, and X3 is N. In some embodiments, X1 is N, X2 is N, and X3 is N.
In some embodiments, R2 is substituted with one or more selected from alkyl, carboxy, carbamate, urea, amide, and halo. In some embodiments, R2 is substituted with carbamate or amide. In some embodiments, R2 is substituted with an alkylcarbamate. In some embodiments, R2 is a group having a structure of:
Figure imgf000005_0001
wherein: n is 0, 1, 2 or 3;
Y1 and Y2 are each independently absent or is O, NR6, or CH2;
Z1 and Z2 are each independently O, N, or C;
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
R6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I, or a pharmaceutically acceptable salt thereof.
In some embodiments, Z1 and Z2 are each independently N or C.
In some embodiments, R2 is a group having a structure of:
Figure imgf000005_0002
wherein: n is 0, 1, 2 or 3;
Y1 is O or CH2;
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
R6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I.
In some embodiments, R2 is a group having a structure of:
Figure imgf000005_0003
wherein: n is 0, 1, 2 or 3;
Y1 is O or CH2; and R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
In some embodiments, R2 is a group having a structure of:
Figure imgf000006_0001
wherein: n is 0, 1, 2 or 3;
Y1 is O or CH2; and
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
Also provided is a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt or prodrug as taught herein. In some embodiments, the composition is formulated for oral or parenteral (e.g. intravenous, intrapleural, intraperitoneal or intraovarian) administration. In some embodiments, the composition is formulated for oral administration and is in the form of a capsule, cachet, lozenge, or tablet. In some embodiments, the formulation is provided in unit dosage form of from 1 mg to 10 grams of the compound, pharmaceutically acceptable salt or prodrug.
Further provided is a method treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof. Also provided is a compound of Formula I or a pharmaceutically acceptable salt or prodrug thereof for use in treating cancer in a subject in need thereof, or for preparing a medicament for use in treating cancer.
In some embodiments, the cancer has PRX3 expression.
In some embodiments, the subject is a human subject. In some embodiments, the subject is a non-human animal subject (e.g. non-human mammalian subject).
In some embodiments, the administering is carried out by administering a pharmaceutical composition comprising said compound or pharmaceutically acceptable salt or prodrug.
In some embodiments, the administering further comprises administering bortezomib, carboplatin, paclitaxel, an immunotherapy agent, or a combination thereof. In some embodiments, the administering further comprises administering doxorubicin. Further provided is a method of inhibiting PRX3 in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof.
DETAILED DESCRIPTION
The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.
The disclosures of all patent references cited herein are hereby incorporated by reference to the extent they are consistent with the disclosure set forth herein. As used herein in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
L Definitions
As used herein in the accompanying chemical structures, "H" refers to a hydrogen atom. "C" refers to a carbon atom. "N" refers to a nitrogen atom. "S" refers to a sulfur atom. "O" refers to an oxygen atom.
Unless indicated otherwise, nomenclature used to describe chemical groups or moieties as used herein follow the convention where, reading the name from left to right, the point of attachment to the rest of the molecule is at the right hand side of the name. For example, the group "alkylamino" is attached to the rest of the molecule at the amino end, whereas the group "aminoalkyl" is attached to the rest of the molecule at the alkyl end.
"Alkyl," as used herein, refers to a saturated straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3 -methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n- nonyl, n-decyl, and the like. "Lower alkyl" as used herein, is a subset of alkyl and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like. The alkyl groups may be optionally substituted with one or more suitable substituents, such as halo, hydroxy, carboxy, amine, etc.
"Cycloalkyl," as used herein, refers to a saturated cyclic hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The cycloalkyl groups may be optionally substituted with one or more suitable substituents, such as halo, hydroxy, carboxy, amine, etc.
"Aryl," as used herein, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused or directly adjoining ring system having one or more aromatic rings. Examples include, but are not limited to, phenyl, indanyl, indenyl, tetrahydronaphthyl, biphenyl, napthyl, azulenyl, etc. The aryl may be optionally substituted with one or more suitable substituents, such as alkyl, halo, hydroxy, carboxy, amine, etc.
"Heteroaryl," as used herein, refers to a monovalent aromatic group having a single ring or two fused or directly adjoining rings and containing in at least one of the rings at least one heteroatom (typically 1 to 3) independently selected from nitrogen, oxygen and sulfur. Examples include, but are not limited to, pyrrole, imidazole, thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, benzothiophene, benzofuran, indole, benzimidazole, benzothiazole, quinoline, isoquinoline, quinazoline, quinoxaline, phenyl -pyrrole, phenyl-thiophene, etc. The heteroaryl may be optionally substituted with one or more suitable substituents, such as alkyl, halo, hydroxy, carboxy, amine, etc.
"Heterocycle" as used herein refers to a saturated or partially unsaturated cyclic hydrocarbon with at least one heteroatom (typically 1 to 3) independently selected from nitrogen, oxygen and sulfur. The heterocycle may be a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. The heterocycle may be optionally substituted with one or more suitable substituents, such as alkyl, halo, hydroxy, carboxy, amine, etc.
"Monocyclic heterocycle" means a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom, and which is not aromatic. Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3- dioxolanyl, dihydropyranyl (including 3, 4-dihydro-2H-pyran-6-yl), 1,3-dithiolanyl, 1,3-dithianyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxadiazolidinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolidinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomor- pholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl.
"Bicyclic heterocycle" means a monocyclic heterocycle fused to an aryl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle. Representative examples of bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3 -benzodi oxolyl, 1,3-benzodithiolyl, 2,3-dihydro-l,4-benzodioxinyl, 2,3 -dihydro-1 -benzofuranyl, 2, 3-dihy dro-1 -benzothienyl, 2,3- dihydro-lH-indolyl, 3,4-dihydroquinolin-2(lH)-one and 1,2, 3, 4- tetrahydroquinolinyl.
"Tricyclic heterocycle" means a bicyclic heterocycle fused to an aryl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle. Representative examples of tricyclic heterocycles include, but are not limited to, 2,3,4,4a,9,9a-hexahydro- IH-carbazolyl, 5a,6,7,8,9,9a-hexahydro- dibenzo[b,d] furanyl, and 5a,6,7,8,9,9a-hexahydrodibenzo[b,d]thienyl.
The terms "halo" and "halogen," as used herein, refer to fluoro (-F), choro (-C1), bromo (- Br), or iodo (-1).
"Haloalkyl" refers to one or more halo groups appended to the parent molecular moiety through an alkyl group. Examples include, but are not limited to, chloromethyl, fluoromethyl, trifluoromethyl, etc.
"Carboxy" refers to the group -COOH.
"Alkoxy" refers to an alkyl or cycloalkyl group, as herein defined, attached to the principal carbon chain through an oxygen atom. Representative examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, and hexyloxy.
"Hydroxy" or "hydroxyl" refers to an -OH group.
An "amine" or "amino" refers to a group -NH2, wherein none, one or two of the hydrogens may be replaced by an alkyl, cycloalkyl, or aryl as defined herein.
An "amide" or "amido" refers to a group having a carbonyl bonded to a nitrogen atom, such as -C(0)NH2, wherein none, one or two of the hydrogens may be replaced by an alkyl, cycloalkyl, or aryl as defined herein.
An "ether" refers to a group in which there is an ether, R-O-R', wherein R and R' are each independently an alkyl, cycloalkyl, or aryl as defined herein.
An "ester" refers to a group in which there is an ester, R-C(O)-O-R', wherein R and R' are each independently an alkyl, cycloalkyl, or aryl as defined herein. A "carbamate" refers to a group in which there is a carbamate, R-O-C(O)NR'R", wherein R, R' and R" are each independently an alkyl, cycloalkyl, or aryl as defined herein.
A "urea" refers to a group in which there is a urea, R-NH-C(O)-NH-R', wherein R and R' are each independently an alkyl, cycloalkyl, or aryl as defined herein.
As understood in the art, the term "optionally substituted" indicates that the specified group is either unsubstituted, or substituted by one or more suitable substituents. A "substituent" that is "substituted" is a group which takes the place of one or more hydrogen atoms on the parent organic molecule.
Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine.
II. Active Compounds
Active compounds useful as PRX3 inhibitors in accordance with the present invention are provided below. Unless otherwise stated, structures depicted herein are also meant to include all enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Tautomeric forms include keto-enol tautomers of a compound. In addition, unless otherwise stated, all rotamer forms of the compounds of the invention are within the scope of the invention.
Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.
Provided herein according to some embodiments as an active compound is a compound of Formula I:
Figure imgf000011_0001
wherein R1 is a 5-, 6-, or 7-membered heteroaryl or a heterocycle containing carbon atoms and at least one heteroatom selected from nitrogen and oxygen, which R1 is substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester and halo; and
A is -(CH2)m-, -0-(CH2)m-, or - (CH2)m-0- , wherein m is 0, 1, 2 or 3, or a pharmaceutically acceptable salt or prodrug thereof.
In some embodiments, R1 is a 5-membered heteroaryl or heterocycle and the compound is a compound of Formula la:
Figure imgf000011_0002
wherein:
A is as defined in claim 1;
X1, X2 and X3 are each independently N or C; and
R2 is an aryl, heteroaryl, cycloalklyl or heterocycle, which R2 is optionally substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester, and halo.
In some embodiments, X1 is N, X2 is C, and X3 is N. In some embodiments, X1 is C, X2 is N, and X3 is N. In some embodiments, X1 is N, X2 is N, and X3 is N.
In some embodiments, R2 is substituted with one or more selected from alkyl, carboxy, carbamate, urea, amide, and halo. In some embodiments, R2 is substituted with carbamate or amide. In some embodiments, R2 is substituted with an alkylcarbamate.
In some embodiments, R2 is a group having a structure of:
Figure imgf000012_0001
wherein: n is 0, 1, 2 or 3;
Y1 and Y2 are each independently absent or is O, NR6, or CH2;
Z1 and Z2 are each independently O, N, or C;
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
R6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I, or a pharmaceutically acceptable salt thereof.
In some embodiments, Z1 and Z2 are each independently N or C.
In some embodiments, R2 is a group having a structure of:
Figure imgf000012_0002
wherein: n is 0, 1, 2 or 3;
Y1 is O or CH2;
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
R6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I.
In some embodiments, R2 is a group having a structure of:
Figure imgf000012_0003
wherein: n is 0, 1, 2 or 3;
Y1 is O or CH2; and R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
In some embodiments, R2 is a group having a structure of:
Figure imgf000013_0001
wherein: n is 0, 1, 2 or 3;
Y1 is O or CH2; and
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I.
Particular examples of active compounds include, but are not limited to, those selected
Figure imgf000013_0002
In some embodiments, an active compound can form a covalent adduct with PRX3 in a biochemical PRX3 inhibition assay, which may support its PRX3 inhibition activity. In some embodiments, an active compound can have an ECso in a cellular activity assay (such as the ability to kill cancer cells such as SKOV3 ovarian cancer cells) in the micromolar range, such as from 0.05, 0.1, 0.25, or 0.5 micromolar, to 10, 15, or 20 micromolar.
In some embodiments, an active compound can have good solubility, e.g., solubility in aqueous solution (e.g., saline such as phosphate buffered saline, water, etc.) of at least 0.1 millimolar, such as from 0.1 to 1 millimolar, or solubility in an aqueous solution of at least 1 millimolar.
In some embodiments, an active compound can have good aqueous stability. For example, in some embodiments, an active compound may have no decrease in purity after 24 hours in an aqueous solution.
In some embodiments, an active compound does not have appreciable antimicrobial activity, e.g., at greater than 20 micromolar concentrations.
In some embodiments, an active compound does not inhibit the proteasome and/or does not inhibit FOXM1 DNA binding. These may indicate that the compound has greater specificity for PRX3 than TS.
III. Methods of Use
The terms "treat", "treatment" and "treating" as used herein refer to any type of treatment that imparts a benefit to a subject afflicted with a disease or disorder, delay in the progression of the disease or disorder, or symptoms thereof, etc. In some embodiments, the treatment is for a cancer (e.g., a cancer having elevated reactive oxygen species).
In some embodiments, the subject treated is a human subject. In some embodiments, the subject is a non-human animal (e.g., non-human mammalian subject). A non-human animal may include, but is not limited to, non-human primates, dogs, cats, horses, cattle, goats, pigs, sheep, guinea pigs, mice, rats and rabbits, as well as any other domestic, commercially or clinically valuable animal, including but not limited to animal models and livestock animals. In some embodiments, the subject is a subject in need of a treatment such as a treatment of the present invention.
Cancers that may be treated with the active compounds according to some embodiments may include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bile duct cancer; bladder cancer; bone cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cardiac tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ductal carcinoma in situ; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; multiple myeloma; heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; histiocytosis; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); melanoma; midline tract carcinoma; multiple endocrine neoplasia syndrome; muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); nasopharynx cancer; neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); parathryroid cancer; papillary adenocarcinoma; penile cancer (e.g., Paget's disease of the penis and scrotum); pharyngeal cancer; pinealoma; pituitary cancer; pleuropulmonary blastoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; retinoblastoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; stomach cancer; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thymic cancer; thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; uterine cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva). See also US 2019/0153098 to Goldberg et al.
In some embodiments, the cancer is a blood cancer such as leukemia, liver cancer, lung cancer, lymphoma, melanoma, prostate cancer, head and neck cancer, bladder cancer, brain cancer, breast cancer, or cervical cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is malignant mesothelioma.
In some embodiments, the cancer has PRX3 expression. For example, the cancer may be a cancer type generally known to express PRX3 and/or the cancer has been determined (e.g., by testing a biopsy) to have PRX3 expression. The cancer may be metastatic, in which cancerous cells from a primary or original tumor migrate to another organ or tissue and may be identified as the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. As a non-limiting example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.
IV. Pharmaceutical formulations
The active compounds disclosed herein can, as noted above, be prepared in the form of their pharmaceutically acceptable salts. Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b) salts formed from elemental anions such as chlorine, bromine, and iodine, and (c) salts derived from bases, such as ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium, and salts with organic bases such as dicyclohexylamine and N-methyl-D-glucamine.
Active compounds of the present invention may be prepared as pharmaceutically acceptable prodrugs. Such prodrugs are those which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable risk/benefit ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term "prodrug" refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Prodrugs as Novel delivery Systems, Vol. 14 of the A.C.S. Symposium Series and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergam on Press, 1987, both of which are incorporated by reference herein. See also US Patent No. 6,680,299. Examples include a prodrug that is metabolized in vivo by a subject to an active drug having an activity of active compounds as described herein, wherein the prodrug is an ester of an alcohol or carboxylic acid group, if such a group is present in the compound; an acetal or ketal of an alcohol group, if such a group is present in the compound; an N-Mannich base or an imine of an amine group, if such a group is present in the compound; or a Schiff base, oxime, acetal, enol ester, oxazolidine, or thiazolidine of a carbonyl group, if such a group is present in the compound, such as described in US Patent No. 6,680,324 and US Patent No. 6,680,322.
The active compounds described above may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (9th Ed. 1995). In the manufacture of a pharmaceutical formulation according to the invention, the active compound (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy comprising admixing the components, optionally including one or more accessory ingredients.
The formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature, severity and location of the condition being treated and on the nature of the particular active compound which is being used.
Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound(s), which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The formulations may be presented in unit/dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising an active compound(s), or a salt thereof, in a unit dosage form in a sealed container. The compound or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt. When the compound or salt is substantially water-insoluble, a sufficient amount of emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.
Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof. Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.
Further, the present invention provides liposomal formulations of the compounds disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol-free. When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques. The liposomal formulations containing the compounds disclosed herein or salts thereof may be lyophilized to produce a lyophilizate which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
Other pharmaceutical compositions may be prepared from the compounds disclosed herein, or salts thereof, such as aqueous base emulsions. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the compound or salt thereof. Particularly useful emulsifying agents include phosphatidyl cholines, and lecithin.
In addition to active compound(s), the pharmaceutical compositions may contain other additives, such as pH-adjusting additives. In particular, useful pH-adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the compositions may contain microbial preservatives. Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use. If desired, the pharmaceutical compositions of the present invention may be lyophilized using techniques well known in the art. V. Dosage and routes of administration.
As noted above, the present invention provides pharmaceutical formulations comprising the active compounds (including the pharmaceutically acceptable salts thereof), in pharmaceutically acceptable carriers for oral, rectal, topical, buccal, parenteral, intrapleural, intraovarian, intramuscular, intradermal, intravascular, and/or transdermal administration. Parenteral administration may be, for example, intravascular (intravenous or intraarterial), intrapleural, intraperitoneal or intraovarian administration by injection, infusion or implantation.
The therapeutically effective dosage of any specific compound, the use of which is in the scope of present invention, will vary somewhat from compound to compound, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg/kg is expected to have therapeutic efficacy, with all weights being calculated based upon the weight of the active compound, including the cases where a salt is employed. Toxicity concerns at the higher level may restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the active base, including the cases where a salt is employed. A dosage from about 10 mg/kg to about 50 mg/kg may be employed for oral administration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg may be employed for intramuscular injection.
Depending upon the condition being treated, the compounds described herein may be administered alone or concurrently with one or more additional active agent useful for treating the disease or condition with which the patient is afflicted. Examples of additional active agents include, but are not limited to, those set forth in paragraphs 0065 through 0387 of W. Hunter, D. Gravett, et al., US Patent Application Publication No. 20050181977 (Published August 18, 2005) (assigned to Angiotech International AG) the disclosure of which is incorporated by reference herein in its entirety.
The present invention is explained in greater detail in the following non-limiting Examples.
EXAMPLES
ABBREVIATIONS mmole: millimoles g: grams mL: milliliters
HOBt.H2O: 1 -hydroxybenzotriazole hydrate
EDC.HC1: 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride HC1: hydrochloric acid
DCM: dichloromethane or methylene chloride
DBU: l,8-diazabicyclo[5.4.0]undec-7-ene
Aq.: aqueous
RT : room temperature
General Synthetic Procedures
Figure imgf000022_0001
C D
General procedures
Example A
The carboxylic acid (1.0 eq.) was dissolved in DCM. L-Serine methyl ester hydrochloride (1.2 eq.) was added, followed by N,N-diisopropylethylamine (2.0 eq.) and a coupling reagent (1.2 eq.). The mixture was stirred at RT until the reaction was complete by TLC analysis. Water (25 mL) was added and the mixture was extracted with DCM (3 x 15 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide Intermediate A.
Example B
Intermediate A was dissolved in DCM. Imidazole (1.2 eq.) was added followed by tertbutyldimethylchlorosilane (1.2 eq.). The reaction was stirred at RT until complete by TLC analysis. Water ((25 mL) was added and the mixture was extracted with DCM (3 x 15 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography. The isolated compound was dissolved in 4/1/1 THF/methanol/water. Lithium hydroxide (3.0 eq.) was added and the mixture was stirred at RT until the reaction was complete as judged by TLC. Water (25 mL) was added and the solution was treated with IN aq. HC1 to pH=3. The mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated to provide Intermediate B.
Example C
Intermediate B (1.0 eq.) was dissolved in DCM and L-serine methyl ester hydrochloride (1.2 eq.) was added. N,N-Diisopropylethylamine (2.0 eq.) was added followed by a coupling reagent (1.2 eq.). The mixture was stirred at RT until the reaction was complete by TLC analysis. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide Intermediate C.
Example D
Intermediate C (1.0 eq.) was dissolved in DCM and cooled to 0°C. Triethylamine (1.5 eq.) was added followed by methanesulfonyl chloride (1.5 eq.). The mixture was stirred at 0° until complete by TLC analysis. Water (25 mL) was added and the mixture was extracted with DCM (3 x 15 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in THF and cooled to 0°C. l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (1.2 eq.) was added and the mixture was stirred at 0° until the reaction was complete by TLC analysis. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide the desired product.
The residue (1.0 eq.) was dissolved in THF. Tetrabutylammonium fluoride (1.1 eq., 1.0M solution in THF) was added and the solution was stirred at RT until the reaction was complete by TLC analysis. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The isolated compound was dissolved in DCM and cooled to 0°C. Triethylamine (1.5 eq.) and methanesulfonyl chloride (1.5 eq.) were added. The mixture was stirred at RT until the reaction was complete by TLC analysis. Water (25 mL) was added and the mixture was extracted with DCM (3 x 15 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in THF and cooled to 0°C. 1,8- Diazabicyclo[5.4.0]undec-7-ene (DBU, 1.2 eq.) was added and the mixture was stirred until complete by TLC analysis. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide the desired product.
Compound Synthesis
Compound 1: Methyl 2-(2-(l-phenyl-lH-imidazole-4-carboxamido)acrylamide)acrylate
Figure imgf000024_0001
Using the general procedure described for Example A, l-phenyl-lH-imidazole-4-carboxylic acid (0.300 g, 1.59 mmole) was converted to methyl (l-phenyl-lH-imidazole-4-carbonyl)-L-serinate (0.652 g).
'H N R (400 MHz, Chloroform-;/) 5 7.92 (d, J= 1.4 Hz, 2H), 7.80 (d, J= 1.4 Hz, 1H), 7.57 - 7.48 (m, 2H), 7.47 - 7.37 (m, 3H), 4.87 (dt, J= 7.8, 3.9 Hz, 1H), 4.17 - 4.02 (m, 2H), 3.82 (s, 3H).
Figure imgf000024_0002
Using the general procedure described for Example B, methyl (l-phenyl-lH-imidazole-4- carbonyl)-L-serinate (0.652 g) was converted to O-(tert-butyldimethylsilyl)-N-(l -phenyl- 1H- imidazole-4-carbonyl)-L-serine (0.380 g, 61%) as a white solid.
’H NMR (400 MHz, Chloroform-^ 5 7.98 (q, J= 1.5 Hz, 2H), 7.81 (d, J= 7.7 Hz, 1H), 7.58 - 7.49 (m, 2H), 7.44 (ddt, J= 8.2, 7.3, 1.2 Hz, 3H), 4.88 - 4.78 (m, 1H), 4.22 (dd, J= 10.1, 3.4 Hz, 1H), 4.02 (dd, J= 10.2, 4.2 Hz, 1H), 0.88 (s, 9H), 0.09 (s, 6H). Methyl 0-( tert-butyldimethylsilyl)-N-( I -phenyl- lH-imidazole-4-carbonyl)-L-seryl-L-serinate (1.3)
Figure imgf000025_0001
Using the general procedure described for Example C, O-(tert-butyldimethylsilyl)-N-(l-phenyl- lH-imidazole-4-carbonyl)-L-serine (0.380 g, 0.976 mmole) was converted to methyl O-(tert- butyldimethylsilyl)-N-(l-phenyl-lH-imidazole-4-carbonyl)-L-seryl-L-serinate (0.376 g, 79%) as a thick, colorless gel.
'H N R (400 MHz, Chloroform-;/) 5 7.93 (d, J= 1.4 Hz, 2H), 7.78 (d, J= 1.4 Hz, 1H), 7.56 - 7.47 (m, 3H), 7.47 - 7.37 (m, 4H), 4.67 (ddd, J= 11.6, 6.8, 3.9 Hz, 2H), 4.19 (dd, J= 9.9, 4.3 Hz, 1H), 4.03 - 3.93 (m, 2H), 3.85 (dd, J= 9.8, 6.3 Hz, 1H), 3.78 (s, 3H), 0.92 (s, 9H), 0.13 (s, 3H), 0.11 (s, 3H).
Figure imgf000025_0002
Using the general procedure for Example D, methyl O-(tert-butyldimethylsilyl)-N-(l-phenyl-lH- imidazole-4-carbonyl)-L-seryl-L-serinate (0.376 g, 0.766 mmole) was converted to methyl 2-(2- (l-phenyl-lH-imidazole-4-carboxamido)acrylamido)acrylate (0.0344 g, 13%) as a white solid. XH NMR (400 MHz, Chloroform-^ 5 9.75 (s, 1H), 8.54 (s, 1H), 7.95 (d, J= 1.4 Hz, 1H), 7.81 (d, J= 1.4 Hz, 1H), 7.58 - 7.47 (m, 2H), 7.43 (tt, J= 7.9, 1.2 Hz, 3H), 6.74 (s, 1H), 6.70 (d, J= 2.1 Hz, 1H), 6.00 (d, J= 1.4 Hz, 1H), 5.43 (t, J= 1.9 Hz, 1H), 3.90 (s, 3H).
Compound 2: Methyl 2-(2-(l-phenyl-lH-pyrazole-4-carboxamido)acrylamido)acrylate
Figure imgf000026_0001
Using the general procedure described for Example A, l-phenyl-lH-pyrazole-4-carboxylic acid (0.300 g, 1.59 mmole) was converted to methyl (l-phenyl-lH-pyrazole-4-carbonyl)-L-serinate as a yellow solid (0.404 g, 88%).
XH NMR (400 MHz, CDCh) 5 8.42 (d, J= 0.8 Hz, 1H), 8.03 (d, J= 0.7 Hz, 1H), 7.74 - 7.64 (m, 2H), 7.52 - 7.43 (m, 2H), 7.40 - 7.31 (m, 1H), 6.87 (d, J= 7.1 Hz, 1H), 4.88 (dt, J= 7.2, 3.6 Hz, 1H), 4.14 - 4.01 (m, 2H), 3.83 (s, 3H).
Methyl O-(tert-butyldimethylsilyl)-N-(l-phenyl-lH-pyrazole-4-carbonyl)-L-serinate (2.2)
Figure imgf000026_0002
Methyl (l-phenyl-lH-pyrazole-4-carbonyl)-L-serinate (0.404 g, 1.40 mmole) was dissolved in DCM (4.2 mL). Imidazole (0.115 g, 1.69 mmole) was added followed by tertbutyldimethylchlorosilane (0.250 g, 1.66 mmole). The mixture was stirred at RT for 24 hours and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide methyl <9-(tert-butyldimethylsilyl)-N-( l - phenyl-lH-pyrazole-4-carbonyl)-Z-serinate as a colorless oil (0.461 g, 82%).
’H NMR (400 MHz, CDCh) 5 8.39 (d, J= 0.7 Hz, 1H), 7.98 (d, J= 0.7 Hz, 1H), 7.76 - 7.66 (m, 2H), 7.54 - 7.45 (m, 2H), 7.40 - 7.32 (m, 1H), 6.65 (d, J= 8.1 Hz, 1H), 4.86 (dt, J= 8.1, 2.9 Hz, 1H), 4.19 - 4.14 (m, 1H), 3.95 (dd, J= 10.1, 3.1 Hz, 1H), 3.79 (s, 3H), 0.89 (s, 9H), 0.05 (s, 6H).
Figure imgf000027_0001
Methyl <9-(tert-butyldimethylsilyl)-7V-(l -phenyl- I H-pyrazole-4-carbonyl)-/.-serinate (0.461 g, 1.14 mmole) was dissolved in 4/1/1 THF/methanol/water (6 mL). Lithium hydroxide (0.081 g, 3.38 mmole) was added and the mixture was stirred at RT for 18 hours. Water (25 mL) was added and the solution was treated with IN aq. HC1 to pH=4. The mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated to provide <9-(tert-butyldimethylsilyl)-/'/-( l -phenyl- l H-pyrazole-4-carbonyl)-/.- serine as a colorless oil (0.300 g) that was carried forward without additional purification.
Figure imgf000027_0002
Using the general procedure described for Example C, < -(tert-butyldimethylsilyl)-7V-(l-phenyl- lH-pyrazole-4-carbonyl)-Z-serine (0.300 g, 0.770 mmole)) was converted to methyl <9-(tert- butyldimethylsilyl)-7V-(l-phenyl-lH-pyrazole-4-carbonyl)-Z-seryl-Z-serinate as a colorless solid (0.167 g, 44%).
’H NMR (400 MHz, CDCh) 5 8.41 (dd, J= 3.2, 0.7 Hz, 1H), 7.99 (t, J= 1.0 Hz, 1H), 7.69 (ddd, J= 8.4, 2.1, 1.0 Hz, 2H), 7.59 (d, J= 7.4 Hz, 1H), 7.48 (ddd, J= 8.5, 7.3, 2.1 Hz, 2H), 7.41 - 7.31 (m, 1H), 6.86 (d, J= 6.1 Hz, 1H), 4.69 (dt, J = 7.4, 3.6 Hz, 1H), 4.64 (ddd, J= 7.9, 3.9, 1.9 Hz, 1H), 4.20 - 4.12 (m, 1H), 4.00 (qd, J= 11.3, 3.6 Hz, 2H), 3.80 (d, J = 3.1 Hz, 3H), 3.79 - 3.73 (m, 1H), 0.93 (s, 9H), 0.15 (s, 3H), 0.13 (s, 3H). Methyl (S)-2-( 3-( ( tert-butyldimethylsilyl)oxy)-2-( I -phenyl-lH-pyrazole-4-
Figure imgf000028_0001
Methyl <9-(tert-butyldimethylsilyl)-7V-(l -phenyl- I H-pyrazole-4-carbonyl )-/.-seryl-/.-serinate (0.167 g, 0.340 mmole) was dissolved in DCM (2 mL) and cooled to 0°C. Triethylamine (0.072 mL, 0.514 mmole) was added followed by methanesulfonyl chloride (0.040 mL, 0.517 mmole). The solution was stirred at 0° for 2 hours and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in THF (2 mL) and cooled to 0°C. 1,8- Diazabicyclo[5.4.0]undec-7-ene (DBU) (0.066 mL, 0.441 mmole) was added and the solution was stirred at 0° for 90 minutes. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide methyl (5)-2-(3-((tert-butyldimethylsilyl)oxy)-2-(l-phenyl-lH- pyrazole-4-carboxamido)propanamido)acrylate as a white solid (0.175 g) that was carried forward without additional purification.
Methyl (S)-2-(3-hydroxy-2-(l-phenyl-lH-pyrazole-4-carboxamido)propanamido)acrylate (2.6)
Figure imgf000028_0002
Methyl (5)-2-(3-((tert-butyldimethylsilyl)oxy)-2-(l -phenyl- lH-pyrazole-4- carboxamido)propanamido)acrylate (0.175 g, 0.370 mmole) was dissolved in THF (2 mL) and tetrabutylammonium fluoride (IM solution in THF, 0.410 mL, 0.410 mmole) was added. The solution was stirred at RT for 20 hours and water (25 mL) was added. The mixture was extracted with ethyl acetate (3 x 10 mL), and the combined organics were dried over magnesium sulfate, filtered, and concentrated to provide methyl (5)-2-(3-hydroxy-2-(l-phenyl-lH-pyrazole-4- carboxamido)propanamido)acrylate as a yellow solid (0.143 g) that was carried forward without additional purification.
Methyl 2-(2-( I -phenyl-lH-pyrazole-4-carboxamido)acrylamido)acrylate (2)
Figure imgf000029_0001
Methyl (S)-2-(3-hydroxy-2-(l-phenyl-lH-pyrazole-4-carboxamido)propanamido)acrylate (0.143 g, 0.399 mmole) was dissolved in DCM (2 mL) and cooled to 0°C. Triethylamine (0.084 mL, 0.599 mmole) was added followed by methanesulfonyl chloride (0.046 mL, 0.594 mmole) and the mixture was stirred at 0° for 2 hours. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in THF and cooled to 0°C. 1,8- Diazabicyclo[5.4.0]undec-7-ene (DBU, 0.078 mL, 0.522 mmole) was added and the solution was stirred at 0° for 90 minutes. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide methyl 2- (2-(l-phenyl-lH-pyrazole-4-carboxamido)acrylamido)acrylate as a white solid (0.0118 g, 9%). XH NMR (400 MHz, CDCh) 5 8.58 (s, 1H), 8.51 (s, 1H), 8.42 (d, J= 0.7 Hz, 1H), 8.10 - 8.02 (m, 1H), 7.78 - 7.66 (m, 2H), 7.56 - 7.44 (m, 2H), 7.42 - 7.32 (m, 1H), 6.74 (d, J= 2.4 Hz, 1H), 6.66 (s, 1H), 6.03 (d, J= 1.3 Hz, 1H), 5.50 - 5.42 (m, 1H), 3.91 (s, 3H).
Compound 3: Tert-butyl 4-(5,8-dimethylene-3,6,9-trioxo-2,10-dioxa-4,7- diazaundecyl)piperidine- 1 -carboxylate
Tert-butyl 4-( ((((2, 5-dioxopyrrolidin-l-yl)oxy)carbonyl)oxy)methyl)piperidine-l -carboxylate
Figure imgf000029_0002
Tert-butyl 4-(hydroxymethyl)piperidine-l -carboxylate (1.001 g, 4.65 mmole) was dissolved in acetonitrile (10 mL). N,N’-Disuccinimidyl carbonate (1.787 g, 7.00 mmole) was added followed by trimethylamine (1.95 mL, 13.9 mmole). The solution was stirred at RT for 3 i hours and was concentrated. The residue was dissolved in ethyl acetate (25 mL) and washed with sat. aq. sodium bicarbonate (1 x 25 mL). The organics were dried over magnesium sulfate, filtered, and concentrated to provide tert-butyl 4-(((((2,5-dioxopyrrolidin-l- yl)oxy)carbonyl)oxy)methyl)piperidine-l -carboxylate as an orange oil (1.972 g) that was carried on without additional purification. tert-butyl (S)-4-( ((( 3-hydroxy-l-methoxy-l-oxopropan-2-yl)carbamoyl)oxy)methyl)piperidine-l- carboxylate (3.2)
Figure imgf000030_0001
L-Serine methyl ester hydrochloride (1.027 g, 6.60 mmole) was dissolved in water (10 mL). Sodium bicarbonate (1.401 g, 16.7 mmole) was added followed by a solution of tert-butyl 4- (((((2,5-dioxopyrrolidin-l-yl)oxy)carbonyl)oxy)methyl)piperidine-l-carboxylate (1.972 g, 5.53 mmole) in 1,4-dioxane (10 mL). The mixture was allowed to stir at RT for 20 hours and water (50 mL) was added. The mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide tert-butyl (S)-4-((((3 -hydroxy- 1 -methoxy- 1- oxopropan-2-yl)carbamoyl)oxy)methyl)piperidine-l -carboxylate as a thick colorless oil (1.215 g, 72% from tert-butyl 4-(hydroxymethyl)piperidine-l -carboxylate) that was carried on without additional purification.
N-( ((1 -(Tert-butoxycarbonyl)piperidin-4-yl)methoxy)carbonyl)-O-( tert-butyldimethylsilyl)-L- serine (3.3)
Figure imgf000031_0001
Using the general procedure described for Example B, tert-butyl (5)-4-((((3 -hydroxy- 1-m ethoxy - l-oxopropan-2-yl)carbamoyl)oxy)methyl)piperidine-l -carboxylate (0.951 g, 2.64 mmole) was converted to N-(((l-(tert-butoxycarbonyl)piperidin-4-yl)methoxy)carbonyl)-O-(tert- butyldimethylsilyl)-L-serine as a colorless oil (1.311 g).
'H NMR (400 MHz, CDCh) 5 5.50 (d, J= 8.3 Hz, 1H), 4.41 (dt, J= 7.8, 3.5 Hz, 1H), 4.12 - 4.07 (m, 2H), 3.95 (dd, J= 8.4, 3.8 Hz, 3H), 3.84 (dd, J= 10.1, 4.0 Hz, 1H), 2.78 - 2.63 (m, 2H), 2.01 - 1.95 (m, 1H), 1.90 - 1.75 (m, 2H), 1.45 (s, 9H), 1.23 - 1.10 (m, 2H), 0.88 (s, 9H), 0.08 - 0.02 (m, 6H).
Tert-butyl 4-( (5S, 8S)-5-( ( tert-butyldimethylsilyl)oxy)methyl)-8-(hydroxymethyl)-3, 6, 9-trioxo-
Figure imgf000031_0002
Using the general procedure described for Example C, 7V-(((l-(tert-butoxycarbonyl)piperidin-4- yl)methoxy)carbonyl)-O-(tert-butyldimethylsilyl)-Z-serine (1.311 g, 2.85 mmole) was converted to tert-butyl 4-((55',85 -5-(((tert-butyldimethylsilyl)oxy)methyl)-8-(hydroxymethyl)-3,6,9-trioxo- 2, 10-dioxa-4,7-diazaundecyl)piperidine-l -carboxylate as a colorless oil (1.040 g, 65%).
XH NMR (400 MHz, CDCh): 5 7.42 (d, J= 13 Hz, 1H), 5.59 (d, J= 6.7 Hz, 1H), 4.65 (dt, J= 7.1, 3.5 Hz, 1H), 4.12 (q, J= 7.2 Hz, 2H), 4.04 (dd, J= 9.9, 4.1 Hz, 1H), 3.97 (td, J= 11.4, 7.1 Hz, 4H), 3.79 (s, 3H), 3.73 (dd, J= 9.9, 6.8 Hz, 1H), 2.76 - 2.63 (m, 2H), 1.45 (s, 9H), 1.17 (qd, J= 12.3, 4.3 Hz, 2H), 0.91 (d, J= 6.2 Hz, 9H), 0.10 (d, J= 2.0 Hz, 6H). Tert-butyl (S)-4-(5-(hydroxymethyl)-8-methylene-3, 6, 9-trioxo-2, 10-dioxa-4, 7-
Figure imgf000032_0001
Tert-butyl 4-((55',85 -5-(((tert-butyldimethylsilyl)oxy)methyl)-8-(hydroxymethyl)-3,6,9-trioxo- 2, 10-dioxa-4,7-diazaundecyl)piperidine-l -carboxylate (0.313 g, 0.557 mmole) was dissolved in DCM (2 mL) and cooled to 0°C. Triethylamine (0.120 mL, 0.856 mmole) was added followed by methanesulfonyl chloride (0.066 mL, 0.853 mmole). The mixture was stirred at 0° for 60 minutes and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in THF (2 mL) and cooled to 0°C. l,8-Diazabicyclo[5.4.0]undec-7-ene (0.130 mL, 0.869 mmole) was added and the solution was stirred at 0° for 90 minutes. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide tert-butyl fS')-4-(5- (((tert-butyldimethylsilyl)oxy)methyl)-8-methylene-3,6,9-trioxo-2,10-dioxa-4,7- diazaundecyl)piperidine-l -carboxylate as a thick, colorless oil (0.282 g, 93%).
Tert-butyl (5)-4-(5-(((tert-butyldimethylsilyl)oxy)methyl)-8-methylene-3,6,9-trioxo-2,10-dioxa- 4,7-diazaundecyl)piperidine-l -carboxylate (0.282 g, 0.519 mmole) was dissolved in THF (1.1 mL). Tetrabutylammonium fluoride (IM solution in THF, 1.05 mL, 1.05 mmole) was added and the mixture was stirred at RT for 3 hours. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide tert-butyl (S)-4-(5-(hydroxymethyl)-8-methylene- 3,6,9-trioxo-2,10-dioxa-4,7-diazaundecyl)piperidine-l-carboxylate as a thick, red, gel (0.230 g, 91%).
1H NMR (400 MHz, CDC13): 5 8.73 - 8.70 (s, 1H), 6.48 (s, 1H), 5.83 (s, 1H), 5.65 - 5.62 (m, 1H), 4.24 - 4.21 (m, 1H), 4.05 - 3.97 (m, 3H), 3.89 - 3.78 (m, 2H), 3.73 (s, 3H), 3.65 - 3.57 (m, 1H), 2.59 - 2.52 (m, 2H), 1.68 - 1.64 (m, 1H), 1.59 - 1.49 (m, 2H), 1.36 (s, 9H), 1.08 - 0.99 (m, 2H). Tert-butyl 4-(5,8-dimethylene-3, 6,9-trioxo-2, 10-dioxa-4, 7-diazaundecyl)piperidine-l-
Figure imgf000033_0001
Tert-butyl (5)-4-(5-(hydroxymethyl)-8-methylene-3,6,9-trioxo-2,10-dioxa-4,7- diazaundecyl)piperidine-l -carboxylate (0.203 g, 0.473 mmole) was dissolved in DCM (2 mL) and cooled to 0°C. Triethylamine (0.100 mL, 0.714 mmole) was added followed by methanesulfonyl chloride (0.056 mL, 0.724 mmole), and the mixture was stirred at 0° for 90 minutes. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried with magnesium sulfate, filtered, and concentrated. The residue was dissolved in THF (2 mL) and cooled to 0°C. l,8-Diazabicyclo[5.4.0]undec-7-ene (0.110 mL, 0.736 mmole) was added and the solution was stirred at 0° for 60 minutes. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide tert-butyl 4-(5,8-dimethylene-3,6,9-trioxo-2,10-dioxa-4,7- diazaundecyl)piperidine-l -carboxylate as a thick, pale yellow gel (0.072 g, 37%).
’H NMR (400 MHz, CDCh): 5 8.48 (s, 1H), 7.43 (s, 1H), 6.62 (s, 1H), 6.18 (s, 1H), 5.98 (d, J= 1.3 Hz, 1H), 5.26 (dd, J= 2.4, 1.6 Hz, 1H), 4.18 - 4.07 (m, 2H), 4.00 (d, J= 6.5 Hz, 2H), 3.89 (s, 3H), 2.70 (t, J = 12.7 Hz, 2H), 1.90 - 1.76 (m, 1H), 1.70 (d, J= 13.1 Hz, 2H), 1.46 (s, 9H), 1.17 (td, J= 12.4, 4.5 Hz, 2H).
Compound 4: Tert-butyl 4-(((3 -((3 -methoxy-3 -oxoprop- l-en-2-yl)amino)-3 -oxoprop- l-en-2- yl)carbamoyl)oxy)piperidine-l -carboxylate
Tert-butyl 4-((((2,5-dioxopyrrolidin-l-yl)oxy)carbonyl)oxy)piperidine-l-carboxylate (4.1)
Figure imgf000033_0002
Tert-butyl 4-hydroxypiperidine-l -carboxylate (1.003 g, 4.98 mmole) was dissolved in acetonitrile (10 mL). Triethylamine (2.10 mL, 15.0 mmole) and N,N’-disuccinimidyl carbonate
(1.702 g, 5.98 mmole) were added and the solution was stirred at RT for 18 hours. The solution was concentrated and the residue was dissolved in ethyl acetate (25 mL). The solution was washed sequentially with sat. aq. sodium bicarbonate (1 x 25 mL) and water (1 x 25 mL), then dried over magnesium sulfate and concentrated to provide tert-butyl 4-((((2,5-dioxopyrrolidin-l- yl)oxy)carbonyl)oxy)piperidine-l -carboxylate as a white solid (1.377 g) that was a 1: 1 mixture with the recovered starting material. The material was carried forward without additional purification.
XH NMR (400 MHz, CDCh): 5 4.98 - 4.94 (m, 1H), 3.82 - 3.76 (m, 1H), 3.47 - 3.41 (m, 2H), 3.30 - 3.24 (m, 2H), 2.79 (s, 4H), 1.98 - 1.90 (m, 1H), 1.84 - 1.73 (m, 2H), 1.47 (s, 9H).
Tert-butyl (S)-4-( ( 3-( tert-butyldimethylsilyl)oxy)-l-methoxy-l-oxopropan-2-
Figure imgf000034_0001
Tert-butyl 4-((((2,5-dioxopyrrolidin-l-yl)oxy)carbonyl)oxy)piperidine-l-carboxylate (1.377 g, 4.02 mmole) was dissolved in DCM (8 mL). L-Serine methyl ester hydrochloride (0.693 g, 4.45 mmole) was added followed by N,N-diisopropylethylamine (0.840 mL, 4.82 mmole) and the solution was stirred at RT for 18 hours. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide tert-butyl (5)-4-(((3 -hydroxy- 1 -methoxy- l-oxopropan-2- yl)carbamoyl)oxy)piperidine-l -carboxylate as a tan oil.
T ert-butyl (5)-4-(((3 -hydroxy- 1 -methoxy- 1 -oxopropan-2-yl)carbamoyl)oxy)piperidine- 1 - carboxylate (1.526 g, 4.41 mmole) was dissolved in DCM (8 mL). Imidazole (0.329 g, 4.83 mmole) was added followed by tert-butyldimethylchlorosilane (0.736 g, 4.88 mmole). The mixture was stirred at RT for 60 minutes and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide tert-butyl (5)-4-(((3 -((tert-butyldimethylsilyl)oxy)- 1 -methoxy- 1 -oxopropan-2- yl)carbamoyl)oxy)piperidine-l -carboxylate as a colorless oil (1.019 g, 44% from tert-butyl 4- hydroxypiperidine- 1 -carboxylate). ’H NMR (400 MHz, CDCh): 5 5.48 (d, J= 8.7 Hz, 1H), 4.83 (tt, J= 8.0, 3.7 Hz, 1H), 4.40 (dt, J = 8.5, 2.8 Hz, 1H), 4.06 (dd, J= 10.1, 2.8 Hz, 1H), 3.84 (dd, J= 10.1, 3.1 Hz, 1H), 3.75 (s, 3H), 3.76 - 3.67 (m, 2H), 3.20 (ddd, J= 13.1, 8.6, 3.6 Hz, 2H), 1.91 - 1.81 (m, 2H), 1.68 - 1.56 (m, 2H), 1.46 (s, 10H), 0.86 (s, 9H), 0.03 (s, 3H), 0.02 (s, 3H).
N-( ((1 -(tert-butoxycarbonyl)piperidin-4-yl)oxy)carbonyl)-O-( tert-butyldimethylsilyl)-L-serine (4.3)
Figure imgf000035_0001
T ert-butyl (5)-4-(((3 -((tert-butyl dimethyl silyl)oxy)- 1 -methoxy- 1 -oxopropan-2- yl)carbamoyl)oxy)piperidine-l -carboxylate (1.019 g, 2.21 mmole) was dissolved in 4/1/1 THF/methanol/water (6 mL). Lithium hydroxide (0.161 g, 6.72 mmole) was added and the mixture was stirred at RT for 3 hours. Water (25 mL) was added and the solution was treated with IN aq. HC1 to pH=4. The mixture was extracted with diethyl ether (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide N- (((l-(tert-butoxycarbonyl)piperidin-4-yl)oxy)carbonyl)-O-(tert-butyldimethylsilyl)-Z-serine as a colorless oil (1.062 g).
’H NMR (400 MHz, CDCh): 5 5.50 (d, J= 8.2 Hz, 1H), 4.83 (dq, J= 4.8, 3.9 Hz, 1H), 4.41 (dd, J= 7.6, 3.8 Hz, 1H), 4.17 - 4.04 (m, 1H), 3.85 (dd, J= 10.1, 3.9 Hz, 1H), 3.74 - 3.63 (m, 2H), 3.21 (ddd, J= 13.1, 8.8, 3.6 Hz, 2H), 1.90 - 1.82 (m, 2H), 1.69 - 1.55 (m, 2H), 1.46 (s, 9H), 0.88 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H).
Tert-butyl 4-( (((6S, 9S)-9-(methoxycarbonyl)-2, 2, 3, 3 -tetramethyl- 7, 12-dioxo-4,l l-dioxa-8-aza-3-
Figure imgf000036_0001
7V-(((l-(Tert-butoxycarbonyl)piperidin-4-yl)oxy)carbonyl)-O-(tert-butyldimethylsilyl)-Z-serine (1.062 g, 2.38 mmole) was dissolved in DCM (5 mL). N,N’ -Carbonyldiimidazole (0.425 g, 2.62 mmole) was added portionwise (gas evolution was observed) and the mixture was stirred at RT for 90 minutes. L-Serine methyl ester hydrochloride (0.407 g, 2.62 mmole) and N,N- diisopropylethylamine (0.500 mL, 2.87 mmole) were added and the mixture was stirred for 18 hours. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in DCM (5 mL). Triethylamine 0.370 mL, 2.64 mmole) and acetic anhydride (0.250 mL, 2.64 mmole) were added and the solution was stirred for 4 hours. Water (50 mL) was added and the mixture was extracted with DCM (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide tert-butyl 4-((((65,95)-9-(methoxycarbonyl)-2,2,3,3-tetramethyl- 7, 12-di oxo-4, 11 -di oxa-8-aza-3-silatridecan-6-yl)carbamoyl)oxy)piperi dine- 1 -carboxylate as a colorless oil (0.733 g).
’H NMR (400 MHz, CDCh): 5 7.24 - 7.13 (m, 1H), 5.72 (d, J= 7.5 Hz, 1H), 4.83 (ddt, J= 15.0, 7.5, 3.8 Hz, 3H), 4.53 (td, J= 10.2, 9.0, 3.5 Hz, 1H), 3.80 (d, J= 5.4 Hz, 3H), 3.77 (s, 1H), 3.75
- 3.63 (m, 2H), 3.20 (dtd, J= 17.0, 7.6, 6.6, 3.7 Hz, 2H), 2.07 (s, 3H), 1.93 - 1.81 (m, 2H), 1.68
- 1.55 (m, 2H), 1.46 (s, 9H), 0.91 (s, 9H), 0.10 (s, 6H). Tert-butyl (S)-4-(((3-hydroxy-l-((3-methoxy-3-oxoprop-l-en-2-yl)amino)-l-oxopropan-2-
Figure imgf000037_0001
Tert-butyl 4-((((65,95)-9-(methoxycarbonyl)-2,2,3,3-tetramethyl-7, 12-dioxo-4, 1 l-dioxa-8-aza-3- silatridecan-6-yl)carbamoyl)oxy)piperidine-l -carboxylate (0.733 g, 1.24 mmole) was dissolved in THF (2.5 mL). Tetrabutylammonium fluoride (IM solution in THF, 3.75 mL, 3.75 mmole) was added and the solution was stirred at RT for 3 hours. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide tert-butyl (S)-4-(((3 -hydroxy- 1 -((3- methoxy-3 -oxoprop- 1 -en-2-yl)amino)- 1 -oxopropan-2-yl)carbamoyl)oxy)piperidine- 1 - carboxylate as a colorless oil (0.552 g).
1H NMR (400 MHz, CDC13): 5 8.42 (s, 1H), 6.59 (s, 1H), 5.94 (s, 1H), 5.87 (d, 1H), 4.90 - 4.79 (m, 1H), 4.41 - 4.28 (m, 1H), 4.20 - 4.14 (m, 1H), 3.81 (s, 3H), 3.75 - 3.59 (m, 3H), 3.25 - 3.19 (m, 2H), 1.91 - 1.83 (m, 2H), 1.70 - 1.57 (m, 2H), 1.47 (s, 9H).
Tert-butyl 4-(((3-(( 3-methoxy-3-oxoprop-l-en-2-yl)amino)-3-oxoprop-l-en-2-
Figure imgf000037_0002
T ert-butyl (S)-4-(((3 -hydroxy- 1 -((3 -m ethoxy-3 -oxoprop- 1 -en-2-yl)amino)- 1 -oxopropan-2- yl)carbamoyl)oxy)piperidine-l -carboxylate (0.552 g, 1.33 mmole)) was dissolved in DCM (2.6 mL)and cooled to 0°C. Triethylamine (0.280 mL, 2.00 mmole) was added followed by methanesulfonyl chloride (0.160 mL, 2.07 mmole), and the mixture was stirred at 0° for 60 minutes. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in THF (2.6 mL) and cooled to 0°C. l,8-Diazabicyclo[5.4.0]undec-7-ene (0.300 mL, 2.01 mmole) was added and the solution was stirred at 0° for 60 minutes. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide tert-butyl 4-(((3 -((3 -m ethoxy-3 -oxoprop- l-en-2-yl)amino)- 3 -oxoprop- l-en-2-yl)carbamoyl)oxy)piperidine-l -carboxylate as a thick, pale yellow oil (0.215 g, 44%).
XH NMR (400 MHz, CDCh): 5 8.48 (s, 1H), 7.43 (s, 1H), 6.63 (s, 1H), 6.18 (s, 1H), 5.99 (d, J= 1.4 Hz, 1H), 5.26 (dd, J= 2.5, 1.5 Hz, 1H), 4.89 (tt, J= 7.8, 3.7 Hz, 1H), 3.89 (s, 3H), 3.68 (dd, J = 13.6, 6.8 Hz, 2H), 3.25 (ddd, J= 13.6, 8.4, 3.7 Hz, 2H), 1.95 - 1.80 (m, 2H), 1.65 (ddd, J= 12.8, 8.3, 3.9 Hz, 2H), 1.46 (s, 9H).
Compound 5: Tert-butyl 4-(4-((3 -((3 -methoxy-3 -oxoprop- l-en-2-yl)amino)-3 -oxoprop- 1-en -2- yl)carbamoyl)- 1H- 1 ,2,3 -tri azol- 1 -yl)piperidine- 1 -carboxylate
Figure imgf000038_0001
Tert-butyl 4-hydroxypiperidine-l -carboxylate (2.001 g, 9.94 mmole) was dissolved in DCM (20 mL) and cooled to 0°C. Triethylamine (1.55 mL, 11.1 mmole) was added followed by methanesulfonyl chloride (0.850 mL, 11.0 mmole). The mixture was stirred at 0° for 90 minutes and water (50 mL) was added. The two layers were separated and the aqueous layer was extracted with DCM (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in DMF (20 mL) and sodium azide (0.841 g, 12.9 mmole) was added. The mixture was heated to 80°C for 20 hours and was cooled to RT. Water (50 mL) was added and the mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in hexane (50 mL) and washed with water (3 x 50 mL). The organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide tert-butyl 4-azidopiperidine-l -carboxylate as a colorless oil (1.657 g, 74%) and the intermediate tert-butyl 4-((methylsulfonyl)oxy)piperidine-l-carboxylate as a white solid (0.231 g, 8%).
’H NMR (400 MHz, CDCh): 5 3.87 - 3.75 (m, 2H), 3.57 (tt, J= 9.0, 3.9 Hz, 1H), 3.09 (ddd, J= 13.3, 9.4, 3.4 Hz, 2H), 1.92 - 1.80 (m, 2H), 1.56 (dp, J= 14.4, 5.2, 4.6 Hz, 2H), 1.46 (s, 9H).
Tert-butyl 4-(4-(ethoxycarbonyl)-lH-l,2, 3-triazol-l-yl)piperidine-l -carboxylate (5.2)
Figure imgf000039_0001
Tert-butyl 4-azidopiperidine-l -carboxylate (1.657 g, 7.32 mmole) was dissolved in acetonitrile (35 mL). Ethyl propiolate (0.750mL, 7.40 mmole) and copper(I) iodide (0.281 g, 1.48 mmole) were added and the mixture was stirred at RT for 3 days. The mixture was concentrated and water (50 mL) was added. The mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide tertbutyl 4-(4-(ethoxycarbonyl)-lH-l,2,3-triazol-l-yl)piperidine-l-carboxylate as a pale yellow solid (2.160 g, 91%).
’H NMR (400 MHz, CDCh): 5 8.10 (s, 1H), 4.68 (tt, J= 11.6, 4.0 Hz, 1H), 4.43 (q, J= 7.1 Hz, 2H), 4.28 (d, J= 51.7 Hz, 2H), 2.94 (t, J= 12.7 Hz, 2H), 2.27 - 2.16 (m, 2H), 2.02 - 1.86 (m, 2H), 1.48 (s, 9H), 1.41 (t, J = 7.1 Hz, 3H).
1-(1 -(Tert-butoxycarbonyl)piperidin-4-yl)-lH-l, 2, 3-triazole-4-carboxylic acid (5.3)
Figure imgf000039_0002
Tert-butyl 4-(4-(ethoxy carbonyl)- \H- 1,2, 3 -triazol- l-yl)piperi dine- 1 -carboxylate (2.160 g, 6.66 mmole) was dissolved in 4/1/1 THF/methanol/water (18 mL). Lithium hydroxide (0.332 g, 13.9 mmole) was added and the mixture was stirred at RT for 3 hours. Water (50 mL) was added and the solution was treated with IN aq. HC1 to pH=4. The mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide l-(l-(tert-butoxycarbonyl)piperidin-4-yl)-U/-l,2,3-triazole-4-carboxylic acid as a pale yellow solid (1.831 g, 93%).
’H NMR (400 MHz, DMSO-t/e): 5 13.06 (s, 1H), 8.79 (s, 1H), 4.76 (tt, J= 11.5, 4.0 Hz, 1H), 4.05 (d, J= 13.0 Hz, 2H), 2.17 - 2.01 (m, 2H), 1.88 (qd, J= 12.2, 4.4 Hz, 2H), 1.42 (s, 9H).
Tert-butyl (S)-4-( 4-( 3-( ( tert-butyldimethylsilyl)oxy)- 1 -methoxy- 1-oxopr opan-2 -y I) carbamoyl) -
Figure imgf000040_0001
l-(l-(Tert-butoxycarbonyl)piperidin-4-yl)-U/-l,2,3-triazole-4-carboxylic acid (1.831 g, 6.18 mmole) was dissolved in DCM (12 mL). Z-Serine methyl ester hydrochloride (1.158 g, 7.44 mmole) was added followed by N,N-diisopropylethylamine (3.20 mL, 18.4 mmole), HOBt.^O (1.141 g, 7.45 mmole) and EDC.HC1 (1.439 g, 7.51 mmole). The mixture was stirred at RT for 4 hours and water (50 mL) was added. The two layers were separated and the aqueous layer was extracted with DCM (2 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in DCM (12 mL) and imidazole (0.477 g, 7.01 mmole) and tert-butyldimethylchlorosilane (1.023 g, 6.79 mmole) were added. The mixture was stirred at RT for 18 hours and water (50 mL) was added. The two layers were separated and the aqueous layer was extracted with DCM (2 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide tert-butyl (5)-4-(4-((3-((tert-butyldimethylsilyl)oxy)-l-methoxy-l- oxopropan-2-yl)carbamoyl)-UT- 1,2, 3 -triazol- 1 -yl)piperi dine- 1 -carboxylate as a colorless oil (1.870 g, 59%). XH NMR (400 MHz, CDCh): 5 8.08 (s, 1H), 7.87 (d, J= 8.6 Hz, 1H), 4.84 (dt, J= 8.6, 3.1 Hz, 1H), 4.64 (tt, J= 11.6, 4.1 Hz, 1H), 4.28 (d, J= 7.7 Hz, 2H), 4.18 (dd, J= 10.1, 2.9 Hz, 1H), 3.92 (dd, J= 10.1, 3.3 Hz, 1H), 3.77 (s, 3H), 2.95 (t, J= 12.9 Hz, 2H), 2.21 (dp, J= 12.1, 2.8, 2.3 Hz, 2H), 2.02 - 1.89 (m, 2H), 1.48 (s, 9H), 0.88 (s, 9H), 0.05 (s, 3H), 0.03 (s, 3H).
N-( 1-(1 -(tert-butoxycarbonyl)piperidin-4-yl)-lH-l, 2, 3-triazole-4-carbonyl)-O-( tertbutyldimethylsilyl) -L-serine (5.5)
Figure imgf000041_0001
Tert-butyl (5)-4-(4-((3-((tert-butyldimethylsilyl)oxy)-l-methoxy-l-oxopropan-2-yl)carbamoyl)- U/-l,2,3-triazol-l-yl)piperidine-l-carboxylate (1.870 g, 3.65 mmole) was dissolved in 4/1/1 THF/methanol/water (12 mL). Lithium hydroxide (0.182 g, 7.60 mmole) was added and the mixture was stirred at RT for 2 hours. Water (50 mL) was added and the solution was treated with IN aq. HC1 to pH=4. The mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide N- ( 1 -(1 -(tert-butoxycarbonyl)piperidin-4-yl)- 1H- 1 ,2,3 -tri azol e-4-carbonyl )-<9-(tert- butyldimethylsilyl)-Z-serine as a white solid (1.689 g, 93%).
1H NMR (400 MHz, CDCh): 5 8.72 - 8.17 (m, 1H), 7.91 (d, J= 8.0 Hz, 1H), 4.81 (tt, J= 7.8, 3.7 Hz, 1H), 4.65 (tt, J= 11.5, 2.9 Hz, 1H), 4.38 - 4.20 (m, 3H), 4.01 - 3.93 (m, 1H), 2.95 (t, J= 12.6 Hz, 2H), 2.28 - 2.13 (m, 2H), 1.94 (s, 2H), 1.48 (s, 9H), 0.89 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H). Tert-butyl 4-( 4-( ( 6S, 9S)-9-(methoxycarbonyl)-2, 2, 3, 3-tetramethyl- 7, 12-dioxo-4, 1 l-dioxa-8-aza-
3-silatridecan-6-yl)carbamoyl)-lH-l,2,3-triazol-l-yl)piperidine-l-carboxylate (5.6)
Figure imgf000042_0001
7V-(l-(l-(Tert-butoxycarbonyl)piperidin-4-yl)-U/-l,2,3-triazole-4-carbonyl)-O-(tert- butyldimethylsilyl)-Z-serine (0.401 g, 0.806 mmole) was dissolved in DCM (1.6 mL). Z-Serine methyl ester hydrochloride (0.151 g, 0.971 mmole) was added followed by N,N- diisopropylethylamine (0.280 mL, 1.61 mmole), HOBt.HzO (0.156 g, 1.02 mmole), and EDC.HC1 (0.189 g, 0.986 mmole). The mixture was stirred at RT for 2 hours and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL) and the combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in DCM (1.6 mL) and triethylamine (0.125 mL, 0.892 mmole) was added followed by acetic anhydride (0.084 mL, 0.889 mmole). The solution was stirred at RT for 18 hours. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide tert-butyl 4-(4-(((65,95)-9-(methoxycarbonyl)-2,2,3,3-tetramethyl- 7, 12-di oxo-4, 11 -dioxa-8-aza-3 -silatridecan-6-yl)carbamoyl)- 1H- 1 ,2,3 -tri azol- 1 -yl)piperidine- 1 - carboxylate as a colorless gel (0.332 g, 64%).
’H NMR (400 MHz, CDCh): 5 8.09 (d, J= 3.1 Hz, 1H), 7.90 (dd, J= 7.2, 4.5 Hz, 1H), 7.37 (d, J = 7.7 Hz, 1H), 4.94 - 4.84 (m, 1H), 4.65 (dtd, J= 11.6, 7.4, 3.8 Hz, 2H), 4.52 - 4.46 (m, 1H), 4.31 (dd, J= 11.4, 3.5 Hz, 3H), 4.22 - 4.15 (m, 1H), 3.83 - 3.77 (m, 1H), 3.76 (s, 3H), 2.95 (t, J = 12.9 Hz, 2H), 2.26 - 2.16 (m, 2H), 2.03 (d, J= 1.0 Hz, 3H), 1.95 (qd, J= 12.1, 4.3 Hz, 2H), 1.48 (s, 9H), 0.93 (s, 9H), 0.16 (s, 6H). Tert-butyl 4-( 4-( 3-( 3-methoxy-3-oxoprop-l-en-2-yl)amino)-3-oxoprop-l-en-2-yl)carbamoyl)-
1H-1, 2, 3-triazol-l-yl)piperidine-l -carboxylate (5)
Figure imgf000043_0001
Tert-butyl 4-(4-(((65,95)-9-(methoxycarbonyl)-2,2,3,3-tetramethyl-7,12-dioxo-4,l l-dioxa-8-aza- 3-silatridecan-6-yl)carbamoyl)-U/-l,2,3-triazol-l-yl)piperidine-l-carboxylate (0.332 g, 0.518 mmole) was dissolved in THF (1 mL). Tetrabutylammonium fluoride (IM solution in THF, 1.20 mL, 1.20 mmole) was added and the mixture was stirred at RT for 90 minutes. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in DCM (2 mL) and cooled to 0°C. Triethylamine (0.088 mL, 0.628 mmole) was added followed by methanesulfonyl chloride (0.050 mL, 0.646 mmole) and the mixture was stirred at 0° for 60 minutes. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in THF (2 mL) and cooled to 0°C. l,8-Diazabicyclo[5.4.0]undec-7-ene (0.093 mL, 0.622 mmole) was added and the solution was stirred at 0° for 45 minutes. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography to provide tert-butyl 4-(4-((3 -((3 -methoxy-3 -oxoprop- l-en-2- yl)amino)-3 -oxoprop- 1 -en-2-yl)carbamoyl)- 1H- 1 ,2,3 -tri azol- 1 -yl)piperidine- 1 -carboxylate as a white solid (0.101 g, 43%).
XH NMR (400 MHz, CDCh): 5 9.66 (s, 1H), 8.51 (s, 1H), 8.11 (s, 1H), 6.71 (s, 1H), 6.68 (d, J= 2.2 Hz, 1H), 6.00 (d, J= 1.3 Hz, 1H), 5.48 (dd, J= 2.3, 1.5 Hz, 1H), 4.66 (tt, J= 11.6, 4.1 Hz, 1H), 4.29 (s, 2H), 3.89 (s, 3H), 2.95 (t, J= 12.8 Hz, 2H), 2.28 - 2.18 (m, 2H), 1.96 (qd, J= 12.2, 4.5 Hz, 2H), 1.49 (s, 9H).
Compound 6: Tert-butyl 4-(3 -((3 -((3 -methoxy-3 -oxoprop- l-en-2-yl)amino)-3 -oxoprop- 1-en -2- yl)carbamoyl)- IH-pyrazol- 1 -yl)piperidine- 1 -carboxylate Tert-butyl 4-( 3-(methoxycarbonyl)-lH-pyr azol- l-yl)piperidine-l -carboxylate (6.1)
Figure imgf000044_0001
Using a previously described procedure (International patent application W02013010453), tertbutyl 4-hydroxypiperidine-l -carboxylate (2.032 g, 10.1 mmole) was dissolved in DCM (20 mL) and cooled to 0°C. Triethylamine (1.55 mL, 11.1 mmole) was added followed by methanesulfonyl chloride (0.850 mL, 11.0 mmole). The mixture was stirred at 0° for 2 hours and water (50 mL) was added. The two layers were separated and the aqueous layer was extracted with DCM (2 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in DMF (20 mL). Methyl IT/-pyrazole-3 -carboxylate (1.530 g, 12.1 mmole) was added followed by potassium carbonate (1.650 g, 11.9 mmole). The mixture was heated to 70°C for 3 days and was cooled to RT. Water (75 mL) was added and the mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography (20-50% ethyl acetate/hexane gradient) to provide tert-butyl 4-(3- (methoxycarbonyl)-lH-pyrazol-l-yl)piperidine-l-carboxylate as a colorless oil (0.582 g, 19%). XH NMR (400 MHz, CDCh): 5 7.51 (d, J= 2.0 Hz, 1H), 6.84 (d, J= 2.0 Hz, 1H), 5.28 (tt, J= 11.4, 4.1 Hz, 1H), 4.23 (d, J = 29.9 Hz, 2H), 3.88 (s, 3H), 3.78 - 3.65 (m, 1H), 3.28 (tdd, J= 13.6, 8.2, 3.7 Hz, 1H), 2.90 (s, 2H), 2.18 - 2.03 (m, 2H), 2.01 - 1.92 (m, 2H). l-( I -(Tert-butoxycarbonyl)piperidin-4-yl)-lH-pyrazole-3-carboxylic acid (6.2)
Figure imgf000044_0002
Tert-butyl 4-(3-(methoxycarbonyl)-lH-pyrazol-l-yl)piperidine-l-carboxylate (0.582 g, 1.88 mmole) was dissolved in 4/1/1 THF/methanol/water (6 mL). Lithium hydroxide monohydrate (0.159 g, 3.79 mmole) was added and the mixture was stirred at RT for 2 hours. Water (25 mL) was added and the solution was treated with IN aq. HC1 to pH=3. The mixture was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide l-(l-(tert-butoxycarbonyl)piperidin-4-yl)-lH-pyrazole-3- carboxylic acid as a colorless oil (0.561 g).
’H NMR (400 MHz, CDCh): 5 7.55 (d, J= 2.0 Hz, 1H), 6.97 (d, J= 2.0 Hz, 1H), 5.26 (tt, J= 11.3, 4.1 Hz, 1H), 4.35 - 4.17 (m, 2H), 2.89 (d, J= 13.3 Hz, 2H), 2.12 (dd, J= 12.1, 3.9 Hz, 2H), 1.98 (d, J= 12.8 Hz, 2H), 1.48 (s, 9H).
Tert-butyl (S)-4-( 3-( 3-( ( tert-butyldimethylsilyl)oxy)-l-methoxy-l-oxopropan-2-yl)carbamoyl)- lH-pyrazol-l-yl)piperidine-l -carboxy late (6.3)
Figure imgf000045_0001
l-(l-(Tert-butoxycarbonyl)piperidin-4-yl)-lH-pyrazole-3-carboxylic acid (0.561 g, 1.90 mmole) was dissolved in DCM (4 mL). L-Serine methyl ester hydrochloride (0.353 g, 2.27 mmole) was added followed by N,N-diisopropylethylamine (0.660 mL, 3.79 mmole), HOBt.^O (0.354 g, 3.21 mmole), and EDC.HC1 (0.441 g, 3.20 mmole). The mixture was stirred at RT for 18 hours and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in DCM (4 mL) and imidazole (0.142 g, 2.09 mmole) and tertbutyldimethylchlorosilane (0.321 g, 2.13 mmole) were added. The mixture was stirred at RT for 3 hours and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography (5-30% ethyl acetate/hexane gradient) to provide tert-butyl (5)-4-(3-((3-((tert-butyldimethylsilyl)oxy)-l-methoxy-l-oxopropan-2- yl)carbamoyl)-U/-pyrazol-l-yl)piperidine-l -carboxylate as a colorless oil (0.636 g, 66%).
’H NMR (400 MHz, CDCh): 5 7.51 (d, J= 2.0 Hz, 1H), 6.80 (d, J= 8.2 Hz, 1H), 6.57 (d, J= 2.0 Hz, 1H), 5.25 (tt, J= 11.4, 4.1 Hz, 1H), 4.76 (dt, J= 8.1, 2.8 Hz, 1H), 4.25 (s, 2H), 4.19 - 4.13 (m, 1H), 3.93 (dd, J= 10.2, 3.1 Hz, 1H), 3.79 (s, 3H), 2.87 (s, 2H), 2.18 - 2.06 (m, 2H), 2.02 - 1.91 (m, 2H), 1.47 (s, 9H), 0.88 (s, 9H), 0.05 (s, 6H).
N-( 1-(1 -(tert-butoxycarbonyl)piperidin-4-yl)-lH-pyrazole-3-carbonyl)-O-( tert-
Figure imgf000046_0001
T ert-butyl (5)-4-(3 -((3 -((tert-butyl dimethyl silyl)oxy)- 1 -methoxy- 1 -oxopropan-2-yl)carbamoyl)- U/-pyrazol-l-yl)piperidine-l -carboxylate (0.636 g, 1.25 mmole) was dissolved in 4/1/1 THF/methanol/water (6 mL). Lithium hydroxide monohydrate (0.107 g, 2.55 mmole) was added and the mixture was stirred at RT for 2 hours. Water (25 mL) was added and the solution was treated with IN aq. HC1 to pH=4. The mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to provide N- (l-(l-(tert-butoxycarbonyl)piperidin-4-yl)-lH-pyrazole-3-carbonyl)-O-(tert-butyldimethylsilyl)- Z-serine as a white solid (0.441 g, 71%). XH NMR (400 MHz, DMSO-tZ): 5 12.87 (s, 1H), 8.54 (d, J= 8.0 Hz, 1H), 7.52 (d, J= 2.0 Hz, 1H), 6.88 (d, J= 2.0 Hz, 1H), 5.33 - 5.19 (m, 1H), 4.51 (td, J= 7.4, 4.9 Hz, 1H), 4.04 (dd, J = 8.8, 4.9 Hz, 2H), 4.00 - 3.86 (m, 2H), 1.92 - 1.77 (m, 4H), 1.41 (s, 9H), 0.83 (s, 9H), 0.04 (s, 3H), 0.02 (s, 3H).
Tert-butyl 4-( 3-( ( 6S, 9S)-9-(methoxycarbonyl)-2, 2, 3, 3-tetramethyl- 7, 12-dioxo-4, 1 l-dioxa-8-aza- 3-silatridecan-6-yl)carbamoyl)-lH-pyrazol-l-yl)piperidine-l -carboxylate ( 6.5)
Figure imgf000047_0001
MCI -( I -(Tert-butoxy carbonyl )pi peri di n-4-y I )- l/7-pyrazol e-3 -carbonyl )-(9-(tert- butyldimethylsilyl)-Z-serine (0.441 g, 0.888 mmole) was dissolved in DCM (1.8 mL). L-Serine methyl ester hydrochloride (0.170 g, 1.09 mmole) was added followed by N,N- diisopropylethylamine (0.310 mL, 1.78 mmole), HOBt.HzO (0.171 g, 1.12 mmole), and EDC.HC1 (0.207 g, 1.08 mmole). The mixture was stirred at RT for 18 hours and water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in DCM (1.8 mL) and triethylamine (0.140 mL, 0.999 mmole) was added, followed by acetic anhydride (0.093 mL, 0.984 mmole). The mixture was stirred at RT for 3 hours. Water (25 mL) was added and the mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography (30-60% ethyl acetate/hexane gradient) to provide tert-butyl 4-(3-(((65,95)-9- (m ethoxy carbonyl)-2, 2, 3, 3 -tetramethyl-7,12-di oxo-4, 11 -di oxa-8-aza-3 -silatridecan-6- yl)carbamoyl)-17/-pyrazol-l -yl)piperidine-l -carboxylate as a colorless oil (0.463 g, 81%). ’H NMR (400 MHz, CDCh): 5 7.50 (d, J= 2.0 Hz, 1H), 7.41 (d, J= 7.7 Hz, 1H), 6.94 (d, J= 6.3 Hz, 1H), 6.58 (t, J= 2.1 Hz, 1H), 5.27 (tt, J= 11.3, 4.1 Hz, 1H), 4.87 (dt, J= 7.6, 3.7 Hz, 1H), 4.57 (ddd, J= 7.8, 6.3, 3.9 Hz, 1H), 4.53 - 4.45 (m, 1H), 4.35 (dd, J= 11.5, 3.4 Hz, 1H), 4.25 (s, 2H), 3.78 (d, J= 1.0 Hz, 3H), 3.74 (ddd, J= 9.9, 7.7, 6.3 Hz, 1H), 2.89 (s, 2H), 2.18 - 2.05 (m, 2H), 2.03 (d, J= 1.2 Hz, 3H), 2.01 - 1.88 (m, 2H), 1.47 (s, 9H), 0.93 (s, 9H), 0.15 (d, J = 1.4 Hz, 3H), 0.14 (s, 3H). Tert-butyl 4-( 3-( 3-( 3-methoxy-3-oxoprop-l-en-2-yl)amino)-3-oxoprop-l-en-2-yl)carbamoyl)- lH-pyrazol-l-yl)piperidine-l -carboxylate ( 6)
Figure imgf000048_0001
Tert-butyl 4-(3-(((65,95)-9-(methoxycarbonyl)-2,2,3,3-tetramethyl-7,12-dioxo-4,l l-dioxa-8-aza- 3 -silatridecan-6-yl)carbamoyl)-lJ/-pyrazol-l-yl)piperidine-l -carboxylate (0.463 g, 7.24 mmole) was dissolved in THF (1.5 mL). TBAF (IM solution in THF, 1.45 mL, 1.45 mmole) was added and the solution was stirred at RT for 2 hours. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in DCM (1.5 mL) and cooled to 0°C. Triethylamine (0.155 mL, 1.11 mmole) and methanesulfonyl chloride (0.084 mL, 1.09 mmole) were added and the mixture was stirred at 0° for 90 minutes. Water (25 mL) was added. The mixture was extracted with DCM (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in THF (1.5 mL) and cooled to 0°C. DBU (0.165 mL, 1.10 mmole) was added and the solution was stirred at 0° for 60 minutes. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography (10-50% ethyl acetate/hexane gradient) to provide tert-butyl 4-(3 -((3 -((3 -methoxy-3 -oxoprop- 1 -en-2-yl)amino)-3 -oxoprop- 1 -en-2- yl)carbamoyl)-U/-pyrazol-l-yl)piperidine-l -carboxylate as a white solid (0.087 g, 27%).
XH NMR (400 MHz, CDCh): 5 8.67 (s, 1H), 8.56 (s, 1H), 7.53 (d, J= 2.1 Hz, 1H), 6.68 (d, J= 2.5 Hz, 1H), 6.66 (d, J= 2.1 Hz, 1H), 6.65 (s, 1H), 6.03 (d, J= 1.3 Hz, 1H), 5.49 (dd, J= 2.4, 1.4 Hz, 1H), 5.27 (tt, J= 11.4, 4.1 Hz, 1H), 4.26 (s, 2H), 3.91 (s, 3H), 2.90 (s, 2H), 2.11 (tt, J= 11.8, 6.3 Hz, 2H), 2.01 (s, 2H), 1.47 (s, 9H).
Compound A (Reference Compound): Methyl 2-(2-(2-(4-(6- bromohexanamido)phenyl)thiazole-4-carboxamido)acrylamido)acrylate
Figure imgf000049_0001
6-Bromohexanoic acid (1.964 g, 10.1 mmole) was dissolved in DCM (20 mL) and DMF (1 drop) was added. Oxalyl chloride (0.880 mL, 10.1 mmole) was added dropwise and the solution was stirred at RT for 90 minutes, then was concentrated. The residue was dissolved in DCM (2 mL) and added dropwise to cold (0°C) solution of 4-(4, 4, 5, 5-tetramethyl-l, 3, 2-dioxaborolan-2- yl)aniline (2.007 g, 9.16 mmole) and N,N-diisopropylethylamine (3.20 mL, 18.4 mmole) in DCM (20 mL). The resulting solution was stirred at 0° for 60 minutes then at RT for 2 hours. Water (50 mL) was added and the two layers were separated. The mixture was extracted with DCM (2 x 20 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography (Isco CombiPrep, 24 g RediSep column, 10-30% ethyl acetate/hexane gradient) to provide 6-bromo-N-(4-(4, 4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)hexanamide as an orange solid (3.241 g, 89%). 1H NMR (400 MHz, Chloroforms/) 5 7.81 - 7.72 (m, 2H), 7.52 (d, J= 8.1 Hz, 2H), 7.17 (s, 1H), 3.42 (t, J= 6.7 Hz, 2H), 2.38 (t, J= 7.4 Hz, 2H), 1.96 - 1.85 (m, 2H), 1.79 (d, J= 7.5 Hz, 2H), 1.57 - 1.48 (m, 2H), 1.34 (s, 12H).
Methyl N-(2-(4-(6-bromohexanamido)phenyl)thiazole-4-carbonyl)-O-(tert-butyldimethylsilyl)-L- ser inate (A.2)
Figure imgf000049_0002
Methyl N-(2-bromothiazole-4-carbonyl)-O-(tert-butyldimethylsilyl)-L-serinate (2.006 g, 4.74 mmole) was dissolved in 1,4-dioxane (9 mL). 6-Bromo-N-(4, 4, 5, 5-tetramethyl-l, 3,2- dioxaborolan-2-yl)phenyl)hexanamide (1.880 g, 4.75 mmole) was added followed by potassium carbonate (2M aqueous solution, 4.70 mL, 9.40 mmole) and bis(triphenylphosphine)palladium(II) chloride (0.332 g, 0.473 mmole). The mixture was heated to 85°C for 18 hours and cooled to RT. Water (50 mL) was added and the mixture was extracted with ethyl acetate (3 x 20 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was purified by silica gel chromatography (Isco CombiPrep, 24 g RediSep column, 20-50% ethyl acetate/hexane gradient) to provide methyl N- (2-(4-(6-bromohexanamido)phenyl)thiazole-4-carbonyl)-O-(tert-butyldimethylsilyl)-L-serinate as a thick, orange gel (1.290 g, 44%).
’H NMR (400 MHz, Chloroform-;/) 5 8.22 (d, J= 8.7 Hz, 1H), 8.05 (s, 1H), 7.94 - 7.85 (m, 2H), 7.67 - 7.60 (m, 2H), 7.51 (s, 1H), 4.90 - 4.81 (m, 1H), 4.22 (dd, J= 10.1, 2.6 Hz, 1H), 3.95 (dd, J= 10.0, 3.4 Hz, 1H), 3.79 (s, 3H), 3.43 (t, J= 6.7 Hz, 2H), 2.41 (t, J= 7.4 Hz, 2H), 1.92 (dq, J = 8.1, 6.8 Hz, 2H), 1.84 - 1.72 (m, 2H), 1.59 - 1.50 (m, 2H), 0.92 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).
N-(2-(4-(6-Bromohexanamido)phenyl)thiazole)-4-carbonyl)-O-(tert-butyldimethylsilyl)-L-serine
(A.3)
Figure imgf000050_0001
Methyl N-(2-(4-(6-bromohexanamido)phenyl)thiazole-4-carbonyl)-O-(tert-butyldimethylsilyl)- L-serinate (1.290 g, 2.11 mmole) was dissolved in 4/1/1 THF/methanol/water (12 mL). Lithium hydroxide (0.158 g, 6.60 mmole) was added and the mixture was stirred at RT for 2 hours. The mixture was poured into water (40 mL) and the solution was treated with IN aq. HC1 to pH=4. The slurry was extracted with ethyl acetate (3 x 15 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated to provide N-(2-(4-(6- bromohexanamido)phenyl)thiazole)-4-carbonyl)-O-(tert-butyldimethylsilyl)-L-serine as an orange foam (1.213 g, 96%).
’H NMR (400 MHz, Chloroform-;/) 5 8.26 (d, J= 8.7 Hz, 1H), 8.05 (s, 1H), 7.84 - 7.81 (m, 2H), 7.74 (s, 1H), 7.67 - 7.60 (m, 2H), 4.90 - 4.81 (m, 1H), 4.26 (dd, J = 10.1, 2.6 Hz, 1H), 3.95 (dd, J= 10.0, 3.4 Hz, 1H), 3.43 (t, J= 6.7 Hz, 2H), 2.41 (t, J= 7.4 Hz, 2H), 1.92 (dq, J= 8.1, 6.8 Hz, 2H), 1.84 - 1.72 (m, 2H), 1.55 - 1.48 (m, 2H), 0.92 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).
Methyl N-(2-(4-(6-bromohexanamido)phenyl)thiazole-4-carbonyl)-O-(tert-butyldimethylsilyl)-L-
Figure imgf000051_0001
Using the procedure described for Example 1.4, N-(2-(4-(6-bromohexanamido)phenyl)thiazole)- 4-carbonyl)-O-(tert-butyldimethylsilyl)-L-serine (1.213 g, 2.03 mmole) was converted to methyl N-(2-(4-(6-bromohexanamido)phenyl)thiazole-4-carbonyl)-O-(tert-butyldimethylsilyl)-L-seryl- L-serinate (1.152 g, 81%) as a yellow gel.
XH NMR (400 MHz, Chloroform-;/) 5 8.24 (d, J= 7.0 Hz, 1H), 8.06 (s, 1H), 7.90 - 7.84 (m, 2H), 7.65 - 7.60 (m, 2H), 7.49 - 7.45 (m, 1H), 7.42 (s, 1H), 4.69 (dq, J= 7.6, 3.8 Hz, 1H), 4.64 (dt, J = 6.7, 3.3 Hz, 1H), 4.25 - 4.16 (m, 1H), 4.05 - 3.93 (m, 2H), 3.89 - 3.81 (m, 1H), 3.79 (s, 3H), 3.44 (t, J= 6.7 Hz, 2H), 2.43 (t, J= 7.4 Hz, 2H), 1.84 - 1.73 (m, 3H), 1.57 (q, J= 8.3 Hz, 3H), 0.95 (s, 9H), 0.14 (s, 3H), 0.14 (s, 3H).
Methyl 2-( 2-( 2-(4-( 6-bromohexanamido)phenyl) thiazole-4-carboxamido)acrylamido)acrylate (A)
Figure imgf000051_0002
Using the procedure described for Compound 2, methyl N-(2-(4-(6- bromohexanamido)phenyl)thiazole-4-carbonyl)-O-(tert-butyldimethylsilyl)-L-seryl-L-serinate (1.151 g, 1.64 mmole) was converted to methyl 2-(2-(2-(4-(6- bromohexanamido)phenyl)thiazole-4-carboxamido)acrylamide)acrylate (0.275 g, 31%) as a white solid.
1H NMR (400 MHz, Chloroform-;/) 6 10.00 (s, 1H), 8.55 (s, 1H), 8.11 (s, 1H), 8.04 - 7.90 (m, 2H), 7.64 (d, J= 8.3 Hz, 2H), 6.78 (d, J= 2.2 Hz, 1H), 6.74 - 6.65 (m, 1H), 6.03 (d, J= 1.2 Hz, 1H), 5.50 (t, J= 1.9 Hz, 1H), 3.91 (s, 3H), 3.44 (t, J= 6.7 Hz, 2H), 2.43 (t, J= 7.4 Hz, 2H), 1.93 (dq, J= 9.1, 6.8 Hz, 2H), 1.80 (p, J= 7.6 Hz, 2H), 1.63 - 1.50 (m, 2H).
Assay Description and Protocols
Description of Activity Assays to Measure PRX3 Inhibition
The compounds described herein generally react with two essential cysteine residues in the mitochondrial peroxidase PRX3, leading to the formation of a non-reducible, covalent crosslink of two PRX3 monomers. This crosslink inactivates PRX3. Increased PRX3 crosslink is associated with mitochondrial stress, increased cell death in cell models of malignant mesothelioma and is associated with decreased tumor volume in a mouse model. Cunniff et al. (2015) PloS one 10, e0127310.
PRX3 uses an essential reduced cysteine residue to reduce hydrogen peroxide. During this process, PRX3 becomes oxidized forming a reversible disulfide bond that links two PRX3 monomers. In the cell, this disulfide can be reduced by the combined activity of thioredoxin 2 (TRX2), thioredoxin reductase 2, and NADPH. The disulfide can also be reduced by small molecule reductants such as dithiolthreitol (DTT). In contrast, the compound-crosslink with PRX3 is irreversible and cannot be broken by the addition of reductants.
To test the activity of compounds, two complimentary assays were used as described below. The Biochemical PRX3 Inhibition Assay tests the ability of each compound to crosslink PRX3 in a simple, in vitro system. In the Cellular Activity Assay, we are testing the ability of compounds to kill SK-OV-3 ovarian cancer cells.
Biochemical PRX3 Inhibition Assay
This assay tests the ability of each compound to form a covalent adduct with PRX3. The assay is performed as described in Nelson et al. (2021) Antioxidants (Basel) 10, 150; and Cunniff et al. (2015) PloS one 10, e0127310, and follows the appearance of non-reducible PRX3 crosslinks by SDS PAGE gel electrophoresis and follows the appearance of single PRX3 adducts by mass spectrometry. For this assay, purified human PRX3, hydrogen peroxide (the PRX3 substrate) and all the components required to enable PRX3 to catalytically cycle (thioredoxin-thioredoxin reductase-NAPDH system) are included (details below). The biochemical PRX3 Inhibition assay contains 100 pM PRX3, 50 pM human TRX2, 0.5 pM mouse thioredoxin reductase, and a NADPH regenerating system composed of 3.2 mM glucose 6-phosphate, 3.2 U/ml glucose 6-phosphate dehydrogenase and 0.4 mM NADPH. Samples are incubated for 1-2 hr at 37°C with either 0.2 mM TS (positive control), compounds or an equivalent volume of DMSO (negative control). During this incubation, hydrogen peroxide is added to induce turnover of PRX3. Reactions are stopped by the addition of a buffer containing 100 mM dithiolthreitol (to break disulfide bonds) and SDS (detergent to denature proteins). NADPH, Glucose 6-Phosphate, and glucose 6-phosphate dehydrogenase were purchased from Sigma Aldrich. PRX3, thioredoxin, and thioredoxin reductase were all purified to >98% purity in the Lowther laboratory according to protocols referenced in Nelson et al. (2021) Antioxidants (Basel) 10, 150; and Cunniff et al. (2015) PloS one 10, e0127310.
To measure the amount of PRX3 crosslink, proteins in the reaction were separated by SDS- polyacrlyamide gel electrophoresis and stained for total protein using GelCode Blue (Life Technologies). The amount of unmodified PRX3 and TS-PRX3 crosslink was measured by densiometric analysis of the signal for the PRX3 band running at the MW of a PRX3 crosslink (~46 kDa) compared to the PRX3 signal at the MW of the un-modified PRX3 (~23 kD).
To measure covalent adduct formation on a single PRX3 monomer, each reaction was exchanged into a mass spectrometry compatible buffer containing 40 mM ammonium citrate, pH 8.3 made in HPLC water. Sample was mixed 1 : 1 with a matrix solution containing 30 mg/mL sinapinic acid in 70% (vol/vol) acetonitrile, 0.2% formic acid and spotted to onto the sample plate. PRX3 mass was measured by MALDLTOF MS analysis on a Bruker Daltonics MALDLTOF MS spectrometer. Spectra were analyzed in FLEXAnalysis Software. The intensity of the reduced PRX3 peak (SH) and intensity of the analog adduct peak was determined and corrected for background signal at the adduct peak in the DMSO control. The fraction of single analog adduct in the monomer peak was determined by dividing the intensity of the adduct peak by the summed intensity of the SH and adduct peaks.
Cellular Activity Assay
The Cellular Activity Assay measures the ECso of each compound in human SK-OV-3 ovarian cancer cells TS to be taken into the cell, transported to the mitochondria, and crosslink PRX3.
The cytotoxicity of compounds was measured as described in Nelson et al. (2021)
Antioxidants (Basel) 10, 150; Cunniff et al. (2015) PloS one 10, e0127310; and Newick et al. (2012) PloS one 7, e39404. SK-OV-3 is an adherent, epithelial, adenocarcenoma cell line obtained from ATCC (ref #: HTB-77). SK-OV-3 cells are resistant to the commonly used chemotherapeutics, cis-platinum and doxorubicin. For this PRX3 crosslinking assay, SK-OV-3 cells were plated in a 96-well plate. After 24 h recovery, the cells were treated for 48 hr with multiple concentrations of each compound ranging from 0.1 - 100 pM.
Cells were washed with PBS to remove dead cells and the remaining live cells were fixed with 3.0% formaldehyde and stained with 0.1% crystal violet. The amount of crystal violet was determined by reading the absorbance at 540 nm (crystal violet dye dissolved in 100% methanol) using a plate reader. The signal for crystal violet dye is proportional to the biomass of remaining live cells. GraphPad Prism9 software was used to calculate the effective inhibitory concentration (ECso) of test compounds. Results for each compound, are normalized to the amount of cells in control wells treated with the equivalent concentration of DMSO (negative control). TS or Compound A (Reference Compound) were included in each set of assays as a positive control.
Solubility Assay
A 20 mM stock solution was made for each compound in DMSO. To measure solubility, 0.03 - 0.05 mL of the stock was added to 0.27 mL 20 mM HEPES, 100 mM NaCl, pH 7.5. The solution was mixed and rotated 18-24 hours at ambient temperature. The next day, solutions were centrifuged for 10 min at 20,000 x g at 24 °C to remove insoluble, solid material. Next, 100 pL of the supernatant containing the soluble compound was added to 900 pL DMSO and High Performance Liquid Chromatography was performed as described in Table 1 and either HPLC gradient 1 (Table 2) for HPLC gradient 2 (Table 3). For each analog, a 200 pM standard and a 20 pM standard were prepared and run in parallel (standards were dissolved completely in 100% DMSO). The amount of compound in the experimental sample was calculated by comparing the area under the curve for compound peak to the area for the closest standard and correcting for dilution.
Table 1. HPLC Instrument Parameters for Solubility Assay
Figure imgf000054_0001
Figure imgf000055_0001
Table 2. HPLC Gradient Program 1
Figure imgf000055_0002
Table 3. Alternate HPLC Gradient 2
Figure imgf000055_0003
Antimicrobial Activity Assay
To determine the minimal inhibitory concentration (MIC) of TS with Enterococcus hirae (ATCC # 10541), a frozen stock of A. hirae was streaked onto a plate containing BHI growth media (7.8 g/L brain extract, 2.0 g/L dextrose, 2.5 g/L di sodium phosphate, 9.7 g/L heart extract, 10 g/L proteose peptone, 5 g/L sodium chloride supplemented with 0.01% (v/v) polysorbate 80, 10 g/mL dextrose, and 10 g/L agar. A single colony of E. hirae was used to inoculate 10 mL of media 41 (described in USP37<81>: 9 g/L pancreatic digest of casein, 5 g/L yeast extract, 20 g/L dextrose, 10 g/L sodium citrate, 1 g/L monobasic potassium phosphate, and 1 g/L dibasic potassium phosphate and grown overnight at 37 °C. The overnight growth was then diluted into growth media to make a single solution of E. hirae with a final optical density at 600 nm = 0.02 AU. A 20 mM stock solution of Thiostrepton in DMSO was made and used to make multiple dilutions of TS in DMSO. Each TS dilution (0.1 mL) was then added to a test tube containing 10 mL of the diluted E. hirae and grown at 37°C overnight while shaking at 180 rpm. The next morning, each tube was visually inspected to determine if the cells grew. Final concentrations tested were 0.120, 0.090, 0.075, 0.060, 0.045, 0.030, and 0.015 pM TS. Complete growth was observed at TS concentrations < 0.030 pM TS and no growth was observed at all TS concentrations > 0.045 pM. Three replicates were made on three separate days (n=9). No intermediate growth was ever observed, and all replicates showed the same growth profile. For TS analogs, the dilute E. hirae was treated with 20 pM final concentration of each analog (three replicates each). No growth inhibition was observed for any analog. DMSO was used as a positive control (complete cell growth) and 20 pM thiostrepton was used as a negative control.
Aqueous Stability Assay
To measure aqueous stability after 24 hours, samples were prepared and analyzed by HPLC as described for the Solubility Assay. % Purity was calculated for the control sample (compound in DMSO) and the assay sample (compound in aqueous buffer) according to the following equations:
Area Compound Peak
% Purity x 100 Total Area of all peaks
Diluent peaks and solvent peaks are excluded from the calculation. Relative stability was calculated by dividing the Purity of the Assay Sample by the Purity of the Standard sample. Assay Results
Table 4 Key
Figure imgf000057_0001
Table 4 Assay Results for Each Compound
Figure imgf000057_0002
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24. Livnat-Levanon, N., Kevei, E., Kleifeld, O., Krutauz, D., Segref, A., Rinaldi, T., Erpapazoglou, Z., Cohen, M., Reis, N., Hoppe, T., and Glickman, M. H. (2014) Reversible 26S proteasome disassembly upon mitochondrial stress. Cell Rep 7, 1371-1380 25. Segref, A., Kevei, E., Pokrzywa, W., Schmeisser, K., Mansfeld, J., Livnat-Levanon, N., Ensenauer, R., Glickman, M. EL, Ristow, M., and Hoppe, T. (2014) Pathogenesis of human mitochondrial diseases is modulated by reduced activity of the ubiquitin/proteasome system. Cell Metab 19, 642-652 26. Ayida, B. K., Simonsen, K. B., Vourloumis, D., and Hermann, T. (2005) Synthesis of dehydroalanine fragments as thiostrepton side chain mimetics. Bioorg Med Chem Lett 15, 2457- 2460
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The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

WHAT IS CLAIMED IS:
1. A compound of Formula I:
Figure imgf000061_0001
wherein R1 is a 5-, 6-, or 7-membered heteroaryl or a heterocycle containing carbon atoms and at least one heteroatom selected from nitrogen and oxygen, which R1 is substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester and halo; and
A is -(CH2)m-, -0-(CH2)m-, or - (CH2)m-0- , wherein m is 0, 1, 2 or 3, or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein R1 is a 5-membered heteroaryl or heterocycle and the compound is a compound of Formula la:
Figure imgf000061_0002
wherein:
A is as defined in claim 1;
X1, X2 and X3 are each independently N or C; and
R2 is an aryl, heteroaryl, cycloalklyl or heterocycle, which R2 is optionally substituted with one or more selected from alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, alkoxy, carboxy, carbamate, urea, amide, amino, ether, ester, and halo, or a pharmaceutically acceptable salt thereof.
3. The compound of claim 2, wherein X1 is N, X2 is C, and X3 is N.
4. The compound of claim 2, wherein X1 is C, X2 is N, and X3 is N.
5. The compound of claim 2, wherein X1 is N, X2 is N, and X3 is N.
6. The compound of any one of claims 2-5, wherein R2 is substituted with one or more selected from alkyl, carboxy, carbamate, urea, amide, and halo.
7. The compound of any one of claims 2-5, wherein R2 is substituted with carbamate or amide.
8. The compound of any one of claims 2-5, wherein R2 is substituted with an alkylcarbamate.
9. The compound of claim 2, wherein R2 is a group having a structure of:
Figure imgf000062_0001
wherein: n is 0, 1, 2 or 3;
Y1 and Y2 are each independently absent or is O, NR6, or CH2;
Z1 and Z2 are each independently O, N, or C;
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
R6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I, or a pharmaceutically acceptable salt thereof.
10. The compound of claim 1, wherein R2 is a group having a structure of:
Figure imgf000062_0002
wherein: n is 0, 1, 2 or 3;
Y1 is O or CH2;
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and
R6 is H or alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein * denotes the connection of the group in the compound of Formula I, or a pharmaceutically acceptable salt thereof.
11. The compound of claim 1, wherein R2 is a group having a structure of:
Figure imgf000063_0001
wherein: n is 0, 1, 2 or 3;
Y1 is O or CH2; and
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I, or a pharmaceutically acceptable salt thereof.
12. The compound of claim 1, wherein R2 is a group having a structure of:
Figure imgf000063_0002
wherein: n is 0, 1, 2 or 3;
Y1 is O or CH2; and
R5 is alkyl (e.g., having from 1 to 8 carbon atoms, linear or branched), wherein said alkyl is optionally substituted (e.g., with halo, amino, ether, alkoxy, or carbamate), or heterocycle; and wherein * denotes the connection of the group in the compound of Formula I, or a pharmaceutically acceptable salt thereof.
13. The compound of claim 1, wherein said compound is selected from the group consisting of:
Figure imgf000064_0001
or a pharmaceutically acceptable salt thereof.
14. A pharmaceutical composition comprising a compound or pharmaceutically acceptable salt of any one of claims 1-13.
15. The composition of claim 14, wherein said composition is formulated for oral or parenteral (e.g. intravenous, intrapleural, intraperitoneal or intraovarian) administration.
16. The composition of claim 15, wherein said composition is formulated for oral administration and is in the form of a capsule, cachet, lozenge, or tablet.
17. The composition of any one of claims 14-16, wherein said formulation is provided in unit dosage form of from 1 mg to 10 grams of the compound, pharmaceutically acceptable salt or prodrug.
18. A method treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutically acceptable salt of any one of claims 1-13.
19. The method of claim 18, wherein the cancer has PRX3 expression.
20. The method of claim 18 or claim 19, wherein said subject is a human subject.
21. The method of claim 18 or claim 19, wherein said subject is a non-human animal subject (e.g. non-human mammalian subject).
22. The method of any one of claims 18-21, wherein said administering is carried out by administering a pharmaceutical composition comprising said compound or pharmaceutically acceptable salt.
23. The method of any one of claims 18-22, wherein said administering further comprises administering bortezomib, carboplatin, paclitaxel, an immunotherapy agent, or a combination thereof.
24. The method of any one of claims 18-23, wherein said administering further comprises administering doxorubicin.
25. A method of inhibiting PRX3 in a subj ect in need thereof, comprising administering to said subject a therapeutically effective amount of a compound or pharmaceutically acceptable salt of any one of claims 1-13.
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